U.S. patent application number 15/534667 was filed with the patent office on 2018-07-05 for contrast agent for imaging hypoxia.
The applicant listed for this patent is OXFORD UNIVERSITY INNOVATION LIMITED. Invention is credited to Benjamin Guy DAVIS, Jurgen SCHNEIDER.
Application Number | 20180185522 15/534667 |
Document ID | / |
Family ID | 54937265 |
Filed Date | 2018-07-05 |
United States Patent
Application |
20180185522 |
Kind Code |
A1 |
DAVIS; Benjamin Guy ; et
al. |
July 5, 2018 |
CONTRAST AGENT FOR IMAGING HYPOXIA
Abstract
The invention relates to a conjugate which comprises an MRI
contrast agent component comprising a nanoparticle, which
nanoparticle comprises a metal or a metal compound, and a hypoxia
targeting moiety which is conjugated to the MRI contrast agent
component, especially wherein said nanoparticle comprises iron
oxide and a biocompatible coating and wherein said hypoxia
targeting moiety is a nitroimidazole. Also provided is the use of a
said conjugate in a method of imaging a subject, a method of
de-testing hypoxia, and a method of evaluating the activity of a
pharmaceutical. Said conjugate is also of use in diagnosis.
Inventors: |
DAVIS; Benjamin Guy;
(Oxford, GB) ; SCHNEIDER; Jurgen; (Oxford,
GB) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
OXFORD UNIVERSITY INNOVATION LIMITED |
OXFORD |
|
GB |
|
|
Family ID: |
54937265 |
Appl. No.: |
15/534667 |
Filed: |
December 11, 2015 |
PCT Filed: |
December 11, 2015 |
PCT NO: |
PCT/GB2015/053870 |
371 Date: |
June 9, 2017 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 49/085 20130101;
A61K 49/1863 20130101; A61K 49/0008 20130101; G01N 33/587
20130101 |
International
Class: |
A61K 49/18 20060101
A61K049/18; A61K 49/08 20060101 A61K049/08; G01N 33/58 20060101
G01N033/58; A61K 49/00 20060101 A61K049/00 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 12, 2014 |
GB |
1422173.3 |
Claims
1. A conjugate which comprises: an MRI contrast agent component
comprising a nanoparticle, which nanoparticle comprises a metal or
a metal compound, and a hypoxia targeting moiety which is
conjugated to the MRI contrast agent component.
2. A conjugate according to claim 1 which comprises a plurality of
said hypoxia targeting moieties conjugated to the MRI contrast
agent component.
3. A conjugate according to claim 1 or claim 2 wherein the or each
hypoxia targeting moiety is conjugated to the MRI contrast agent
component via a linker group.
4. A conjugate according to claim 3 wherein the or each hypoxia
targeting moiety is covalently bonded to the MRI contrast agent
component via said linker group.
5. A conjugate according to any one of the preceding claims wherein
the or each hypoxia targeting moiety comprises a heteroaryl group,
which heteroaryl group is substituted with a nitro group and is
otherwise unsubstituted or substituted.
6. A conjugate according to any one of the preceding claims wherein
the or each hypoxia targeting moiety comprises an imidazolyl group,
which imidazolyl group is substituted with a nitro group and is
otherwise unsubstituted or substituted.
7. A conjugate according to any one of the preceding claims wherein
the or each hypoxia targeting moiety is an unsubstituted or
substituted 2-nitroimidazolyl group which is: a group of formula
(I) ##STR00008## or a pharmaceutically acceptable salt thereof;
wherein: one of R.sup.1, R.sup.2 and R.sup.3 is a bond attaching
the hypoxia targeting moiety to the MRI contrast agent component or
to a linker group which is bonded to the MRI contrast agent
component; and the other two of R.sup.1, R.sup.2 and R.sup.3, which
are the same or different, are independently selected from H, OH,
halo, unsubstituted or substituted C.sub.1-10 alkyl, unsubstituted
or substituted C.sub.2-10 alkenyl, unsubstituted or substituted
C.sub.2-10 alkynyl, unsubstituted or substituted aryl,
unsubstituted or substituted heteroaryl, unsubstituted or
substituted C.sub.3-10 cycloalkyl, unsubstituted or substituted
C.sub.3-10 heterocyclyl, unsubstituted or substituted C.sub.1-10
alkoxy, unsubstituted or substituted aryloxy, --N(R.sup.4).sub.2,
--NO.sub.2, SR.sup.4, --SO.sub.2R.sup.4, --CN, --C(O)R.sup.4,
--OC(O)R.sup.4, --C(O)OR.sup.4, --C(O)N(R.sup.4).sub.2,
--NR.sup.4C(.dbd.O)R.sup.4 and --OP(O)(OR.sup.4).sub.2, provided
that, when the other two of R.sup.1, R.sup.2 and R.sup.3 are at
adjacent positions on the imidazolyl ring, said other two of
R.sup.1, R.sup.2 and R.sup.3 may together form an unsubstituted or
substituted C.sub.2-4 alkylene group; and the or each R.sup.4 is
independently selected from H, unsubstituted or substituted
C.sub.1-6 alkyl, unsubstituted or substituted C.sub.2-6 alkenyl,
unsubstituted or substituted C.sub.2-6 alkynyl, unsubstituted or
substituted aryl, unsubstituted or substituted heteroaryl,
unsubstituted or substituted C.sub.3-10 cycloalkyl, and
unsubstituted or substituted C.sub.3-10 heterocyclyl.
8. A conjugate according to claim 7 wherein one of R.sup.1, R.sup.2
and R.sup.3 is a bond attaching the hypoxia targeting moiety to a
linker group which is bonded to the MRI contrast agent component,
and the other two of R.sup.1, R.sup.2 and R.sup.3 are as defined in
claim 7.
9. A conjugate according to claim 7 wherein R.sup.3 is a bond
attaching the hypoxia targeting moiety to a linker group which is
bonded to the MRI contrast agent component, and R.sup.1 and R.sup.2
which are the same or different, are independently selected from H,
OH, halo, unsubstituted or substituted C.sub.1-10 alkyl,
unsubstituted or substituted C.sub.2-10 alkenyl, unsubstituted or
substituted C.sub.2-10 alkynyl, unsubstituted or substituted aryl,
unsubstituted or substituted heteroaryl, unsubstituted or
substituted C.sub.3-10 cycloalkyl, unsubstituted or substituted
C.sub.3-10 heterocyclyl, unsubstituted or substituted C.sub.1-10
alkoxy, unsubstituted or substituted aryloxy, --N(R.sup.4).sub.2,
--NO.sub.2, SR.sup.4, --SO.sub.2R.sup.4, --CN, --C(O)R.sup.4,
--OC(O)R.sup.4, --C(O)OR.sup.4, --C(O)N(R.sup.4).sub.2,
--NR.sup.4C(.dbd.O)R.sup.4 and --OP(O)(OR.sup.4).sub.2, provided
that R.sup.1 and R.sup.2 may together form an unsubstituted or
substituted C.sub.2-4 alkylene group; and the or each R.sup.4 is as
defined in claim 7.
10. A conjugate according to claim 9 wherein R.sup.1 and R.sup.2
are independently selected from H, OH, halo, NH.sub.2, and
unsubstituted or substituted C.sub.1-3 alkyl.
11. A conjugate according to claim 9 wherein R.sup.1 and R.sup.2
are both H.
12. A conjugate according to any one of claims 3, 4 and 7 to 11
wherein the linker group is a group of formula (II) ##STR00009##
wherein * is the point of attachment of the group of formula (II)
to the hypoxia targeting moiety; X is NR.sup.5 or O; L.sup.1 is
unsubstituted or substituted C.sub.1-10 alkylene which C.sub.1-10
alkylene is optionally interrupted by N(R.sup.5), O, S, C(O),
C(O)N(R.sup.5), N(R.sup.5)C(O), OC(O), C(O)O, arylene or
heteroarylene; n is 0 or an integer of from 1 to 3; the or each Y
is independently selected from S, N(R.sup.5), O, C(O),
C(O)N(R.sup.5), N(R.sup.5)C(O), OC(O), C(O)O, C(R.sup.5).sub.2,
arylene and heteroarylene; L.sup.2 is unsubstituted or substituted
C.sub.1-10 alkylene which C.sub.1-10 alkylene is optionally
interrupted by N(R.sup.5), O, S, C(O), C(O)N(R.sup.5),
N(R.sup.5)C(O), OC(O), C(O)O, arylene or heteroarylene; Z is a bond
attaching the group of formula (II) to the MRI contrast agent
component; and the or each R.sup.5 is independently selected from
H, unsubstituted or substituted C.sub.1-6 alkyl, unsubstituted or
substituted C.sub.2-6 alkenyl, unsubstituted or substituted
C.sub.2-6 alkynyl, unsubstituted or substituted aryl, and
unsubstituted or substituted heteroaryl.
13. A conjugate according to claim 12 wherein X is NH; L.sup.1 is
CH.sub.2; n is 0 or 1; Y is S; and L.sup.2 is unsubstituted
C.sub.1-10 alkylene which is optionally interrupted by N(R.sup.5),
O, S, C(O), C(O)N(R.sup.5), N(R.sup.5)C(O), OC(O) or C(O)O, wherein
R.sup.5 is as defined in claim 12.
14. A conjugate according to claim 12 or claim 13 wherein n is
0.
15. A conjugate according to any one of claims 12 to 14 wherein Z
is a bond attaching the group of formula (II) to a functional group
in the MRI contrast agent component.
16. A conjugate according to claim 15 wherein the functional group
is an amine group and Z is a bond attaching the group of formula
(II) to the nitrogen atom of the amine group.
17. A conjugate according to any one of the preceding claims
wherein said nanoparticle comprises (a) said metal or metal
compound, and (b) a biocompatible coating, wherein the
biocompatible coating comprises a plurality of functional groups,
wherein the or each linker group is bonded to a said functional
group.
18. A conjugate according to any one of claims 12 to 14 wherein
said nanoparticle comprises (a) said metal or metal compound, and
(b) a biocompatible coating, wherein the biocompatible coating
comprises a plurality of functional groups, wherein Z is a bond
attaching the group of formula (II) to a said functional group.
19. A conjugate according to claim 18 wherein the functional groups
are amine groups, and Z is a bond attaching the group of formula
(II) to the nitrogen atom of a said amine group.
20. A conjugate according to claim 19 which comprises a plurality
of said hypoxia targeting moieties, each of which is covalently
bonded to the MRI contrast agent component by a said linker group
of formula (II), wherein Z in each linker group is a bond attaching
the group of formula (II) to the nitrogen atom of a said amine
group.
21. A conjugate according to claim 20 wherein at least 10% of the
amine groups of the biocompatible coating are attached to a group
of formula (II).
22. A conjugate according to claim 20 or claim 21 wherein at least
30% of the amine groups of the biocompatible coating are attached
to a group of formula (II).
23. A conjugate according to any one of claims 17 to 22, wherein
the biocompatible coating comprises a carbohydrate, a sugar, a
sugar alcohol, poly(ethylene glycol) (PEG), a nucleic acid, an
amino acid, a peptide or a lipid.
24. A conjugate according to any one of claims 17 to 23, wherein
the biocompatible coating comprises dextran.
25. A conjugate according to any one of claims 17 to 24, wherein
the biocompatible coating comprises crosslinked dextran.
26. A conjugate according to claim 24 or claim 25 wherein the
dextran is amine-functionalised dextran, which amine-functionalised
dextran comprises a plurality of amine groups.
27. A conjugate according to claim 26 wherein said
amine-functionalised dextran is obtainable by treating dextran with
epichlorohydrin and ammonia.
28. A conjugate according to any one of the preceding claims
wherein said metal or metal compound is ferromagnetic or
ferrimagnetic.
29. A conjugate according to any one of the preceding claims
wherein said nanoparticle is superparamagnetic.
30. A conjugate according to any one of the preceding claims
wherein said metal or metal compound is capable of accelerating the
dephasing of protons in an MRI experiment by shortening the T.sub.2
and T.sub.2* relaxation times.
31. A conjugate according to any one of the preceding claims
wherein: said metal is iron, gadolinium, manganese, cobalt or
nickel, or an alloy comprising iron, gadolinium, manganese, cobalt
or nickel and one or further metals; and said metal compound is an
oxide of iron, a mixed oxide of iron and another metal, or an oxide
of chromium.
32. A conjugate according to any one of the preceding claims
wherein the nanoparticle comprises said metal compound, which metal
compound is an oxide of iron or a mixed oxide of iron and another
metal
33. A conjugate according to claim 32 wherein the metal compound is
iron oxide.
34. A conjugate according to claim 33 wherein the iron oxide
comprises Fe.sub.2O.sub.3 or Fe.sub.3O.sub.4.
35. A conjugate according to any one of the preceding claims
wherein: the nanoparticle comprises (a) iron oxide, and (b) a
biocompatible coating comprising amine-functionalised dextran,
which amine-functionalised dextran comprises a plurality of amine
groups; and the conjugate comprises a plurality of said hypoxia
targeting moieties, each of which is a 2-nitroimidazolyl group, and
each of which is covalently bonded, via a linker group, to the
nitrogen atom of a different one of said amine groups, wherein each
hypoxia targeting moiety and linker group together form a group of
formula (III) ##STR00010## wherein * is the point of attachment of
the group of formula (III) to the nitrogen atom of one of said
amine groups.
36. A conjugate according to claim 35 wherein at least 10% of the
amine groups of the biocompatible coating are attached to a group
of formula (III).
37. A conjugate according to claim 35 or claim 36 wherein at least
30% of the amine groups of the biocompatible coating are attached
to a group of formula (III).
38. A composition comprising a conjugate as defined in any one of
the preceding claims and a pharmaceutically acceptable
excipient.
39. A contrast agent which comprises: a conjugate as defined in any
one of claims 1 to 37 or a composition as defined in claim 38.
40. Use of a conjugate as defined in any one of claims 1 to 37, or
of a composition as defined in claim 38, as a contrast agent.
41. Use of a conjugate as defined in any one of claims 1 to 37, or
of a composition as defined in claim 38, as a contrast agent for
detecting hypoxia.
42. Use of a conjugate as defined in any one of claims 1 to 37, or
of a composition as defined in claim 38, as an MRI contrast
agent.
43. A method of imaging a subject, which method comprises: (a)
administering to the subject a conjugate as defined in any one of
claims 1 to 37, a composition as defined in claim 38, or a contrast
agent as defined in claim 39; and (b) imaging the subject.
44. A method according to claim 43 which comprises (b) imaging the
subject by MRI.
45. A method of detecting hypoxia in a subject, which method
comprises: (a) administering to the subject a conjugate as defined
in any one of claims 1 to 37, a composition as defined in claim 38,
or a contrast agent as defined in claim 39; and (b) detecting the
conjugate in the subject by MRI.
46. A method according to claim 45 wherein detecting the conjugate
by MRI comprises imaging the myocardium.
47. A method according to claim 45 or 46 wherein the subject (i)
has suffered from or is suffering from cardiac arrest, or (ii) is
susceptible to cardiac arrest.
48. A method according to claim 45 wherein detecting the conjugate
by MRI comprises imaging a cancerous, pre-cancerous or benign
tumour or growth.
49. A method according to claim 45 or 48 wherein the subject (i)
has suffered from or is suffering from cancer, or (ii) is
susceptible to cancer.
50. A method according to any one of claims 43 to 49 wherein the
subject is a mammal.
51. A method according to any one of claims 43 to 50 wherein the
subject is a human.
52. An in vitro method of imaging a cell or tissue sample, which
method comprises: (a) contacting the cell or tissue sample with a
conjugate as defined in any one of claims 1 to 37, a composition as
defined in claim 38, or a contrast agent as defined in claim 39;
and (b) imaging the cell or tissue sample.
53. A method according to claim 52 which comprises (b) imaging the
cell or tissue sample by MRI.
54. A conjugate as defined in any one of claims 1 to 37, a
composition as defined in claim 38, or a contrast agent as defined
in claim 39, for use in a diagnostic method practised on the human
or animal body.
55. A conjugate, composition or contrast agent as claimed in claim
54, for use in a diagnostic method practised on the human or animal
body for diagnosing a disease or condition associated with
hypoxia.
56. A conjugate, composition or contrast agent as claimed in claim
55, wherein the disease or condition associated with hypoxia is
cancer, ventricular fibrillation, coronary heart disease,
cardiomyopathy, congenital heart disease, heart valve disease,
acute myocarditis or Long QT Syndrome.
57. A conjugate as defined in any one of claims 1 to 37, a
composition as defined in claim 38, or a contrast agent as defined
in claim 39, for use in a method as defined in any one of claims 43
to 51.
58. A method of evaluating the activity of a pharmaceutical, which
method comprises: (vi) administering to a subject a conjugate as
defined in any one of claims 1 to 37, a composition as defined in
claim 38, or a contrast agent as defined in claim 39; (vii)
detecting the conjugate in the subject by MRI prior to
administering the pharmaceutical to the subject; (viii)
administering the pharmaceutical to the subject; (ix) detecting the
conjugate in the subject by MRI after administering the
pharmaceutical to the subject; and (x) evaluating changes in the
MRI response of the conjugate before and after administration of
the pharmaceutical.
59. A method according to claim 58 wherein the pharmaceutical is
for the treatment, prevention or suppression of a disease or
condition associated with hypoxia.
60. A method according to claim 59 wherein the disease or condition
associated with hypoxia is cancer, ventricular fibrillation,
coronary heart disease, cardiomyopathy, congenital heart disease,
heart valve disease, acute myocarditis or Long QT Syndrome.
61. A method according to any one of claims 58 to 60 wherein the
subject is an animal model for cardiac arrest or cancer.
Description
FIELD OF THE INVENTION
[0001] The invention relates to a conjugate and to the use of the
conjugate as a contrast agent, particularly for imaging hypoxia by
Magnetic Resonance Imaging (MRI).
BACKGROUND OF THE INVENTION
[0002] Tissue hypoxia occurs in pathologic conditions, such as
cancer, ischemic heart disease (when an artery is occluded) and
stroke when oxygen demand is greater than oxygen supply (Sinusas,
A. J. The Potential for Myocardial Imaging with Hypoxia Markers.
Seminars in Nuclear Medicine, XXIX(4), 330-338, 1999). Currently,
most non-invasive approaches to detect hypoxia, for example in the
myocardium, measure either the level of myocardial oxygen supply or
the downstream effects of myocardial hypoxia such as altered
mechanical function or electric instability. However, to date the
direct imaging of the hypoxic myocardium has remained challenging.
Techniques using misonidazole positron emission tomography
(PET)-tracers have been investigated, but these techniques require
exposure to ionizing radiation, which is problematic for in vivo
studies.
[0003] Magnetic Resonance (MR) Imaging is one of the most powerful
diagnosis tools in medical science, and is the preferred method for
imaging the brain and central nervous system, assessing cardiac
function and detecting tumors (Na, H. B.; Hyeon, T. Inorganic
particles for MRI Contrast Agents. Adv. Mater., 21, 2133-2148,
2009; Teja, Amyn S. et al., Synthesis, properties and applications
of magnetic iron oxide nanoparticles, Progress in Crystal Growth
and Characterization of Materials 55:22, 2009). Magnetic resonance
imaging (MRI) relies on exposing a sample to an magnetic field
which results in a perturbation in the magnetic moment exerted by
the spin of nuclei such as the proton. Following the exposure of
the sample to the magnetic field, the spin states of the nuclei
relax to their equilibrium values. The rate of this relaxation
provides information on the nature and environment of the nuclei,
for example allowing MRI to detect differences between tissue
types. However, MRI can yield images that may not be accurate. For
example, in some cases normal tissues show small differences in
relaxation properties when compared to some lesions, leading to
ambiguities in the images that are generated by MRI.
[0004] Nitroimidazoles are a class of compounds comprising an
imidazole ring and a nitro group. Nitroimidazoles have been shown
to have some therapeutic activities. For example, nitroimidazoles
have been shown to display antiparasitic and anti-inflammatory
properties.
[0005] Nitroimidazoles have been shown to be metabolised in an
oxygen-sensitive manner. When cells are deprived of oxygen ("oxygen
starved"), nitroimidazoles undergo a different intracellular
metabolism pathway to that experienced in normal oxygen levels
(McCleland, R. A. et al., R. Biochem. Pharmacol., 33, 303-309,
1985; S. Monge et al., Tetrahedron 57, 9979-9987, 2001). One aspect
of this is that nitroimidazoles have been shown to accumulate in
areas of hypoxia.
[0006] Although the mechanism by which this occurs is not well
known, and without being bound by theory, it is believed that
nitroimidazoles passively diffuse across the cell membrane. Once in
the cytoplasm of the cell, reduction occurs with formation of the
radical anion (R--NO.sub.2). These processes occur in an oxygen
concentration-independent manner. In normoxic conditions, the
radical anion interacts with oxygen in the cell, yielding
superoxide and non-charged misonidazole. The non-charged
misonidazole then diffuses back out of the cell. In contrast, under
hypoxic conditions, a further reduction of the radical anion
occurs, yielding nitroso compounds and hydroxylamines (D. I.
Edwards, Nitroimidazole drugs--action and resistance mechanisms.
Antimicrob. Chemother., 31, 9-20, 1993). This reduction produces
reactive intermediates that bind with cell components of hypoxic
tissues.
[0007] Based on this property, various hypoxic tracers containing
nitroimidazoles have been synthesized, such as
[.sup.18F]-fluoroazomycin arabinoside,
[.sup.18F]-fluoromisonidazole ([.sup.18F]-F-MISO) or (EF-5)
(Sharma, R., Current Radiopharmaceuticals, 4 (4), 2011; Y. Joyard
et al. Bioorg. Med. Chem., 21, 3680-3688, 2013). However, the
interpretation of images obtained using these compounds as contrast
agents can be inaccurate, due to the low signal to noise ratio
related to the tracer itself.
[0008] There is therefore a need for highly specific MR-sensitive
markers of hypoxia, which can be indirectly picked-up by
conventional MRI techniques. There is also a need for substances
which function as MRI contrast agents and which can be used to
detect and to quantify the area of myocardium exposed to ischemia
during a heart attack, and therefore at risk following
reperfusion.
SUMMARY OF THE INVENTION
[0009] The inventors have thus developed a conjugate for use as an
MRI contrast agent, which is of particular use in the imaging of
hypoxia. Specifically, the conjugate overcomes many of the problems
associated with known contrast agents. For example, use of the
conjugate as an MRI contrast agent does not require exposure of a
subject to ionizing radiation. Said use is also advantageous as the
relaxation properties of normal (normoxic) tissues significantly
differ from those of hypoxic tissues such as lesions when the
conjugate is used. The images thus obtained therefore avoid many of
the problems associated with the inherently similar relaxation
times of lesions and normal tissues. Images obtained using the
conjugate as a contrast agent are also advantageous due to the high
signal to noise ratio obtained. Specifically, the conjugate is able
to shorten T.sub.2 and T.sub.2* relaxation times, which leads to a
decreased signal intensity and dark contrast in MRI. This enables
highly sensitive tracking with very low detection limits. Also, the
comparatively large size of the MRI contrast agent component in the
conjugate allows for its efficient conjugation with multiple
biotargeting probes such as hypoxia targeting moieties.
[0010] Accordingly, in one aspect the invention provides a
conjugate which comprises an MRI contrast agent component
comprising a nanoparticle, which nanoparticle comprises a metal or
a metal compound, and a hypoxia targeting moiety which is
conjugated to the MRI contrast agent component. Usually, a
plurality of hypoxia targeting moieties are conjugated to the MRI
contrast agent component, and the or each hypoxia targeting moiety
is typically conjugated to the MRI contrast agent component via a
linker.
[0011] The conjugate finds use in medical applications. Thus, the
invention further provides a composition comprising the conjugate
of the invention and a pharmaceutically acceptable excipient.
Further provided is a contrast agent comprising the conjugate of
the invention or the composition of the invention. Still further
provided is the use of the conjugate of the invention, or of the
composition of the invention, as a contrast agent. The contrast
agent may be a contrast agent for detecting hypoxia, or an MRI
contrast agent.
[0012] The conjugate finds particular use in imaging methods.
Therefore, in yet another aspect, the invention provides a method
of imaging a subject, which method comprises: (a) administering to
the subject the conjugate of the invention, the composition of the
invention, or the contrast agent of the invention; and (b) imaging
the subject.
[0013] The imaging techniques can be used to detect medically
relevant conditions. Therefore, in another aspect, the invention
provides a method of detecting hypoxia in a subject, which method
comprises: (a) administering to the subject the conjugate of the
invention, the composition of the invention, or the contrast agent
of the invention; and (b) detecting the conjugate in the subject by
MRI. For example, detecting the conjugate may comprise imaging the
myocardium, or, for instance, detecting the conjugate may comprise
imaging a cancerous, pre-cancerous or benign tumour or growth.
[0014] The conjugate of the invention not only finds use in in vivo
applications, but also in in vitro methods. Accordingly, the
invention further provides an in vitro method of imaging a cell or
tissue sample, which method comprises: (a) contacting the cell or
tissue sample with the conjugate of the invention, the composition
of the invention, or the contrast agent of the invention; and (b)
imaging the cell or tissue sample.
[0015] Detecting conditions such as hypoxia is of relevance in
medical diagnosis. Thus, in another aspect, the invention provides
the conjugate of the invention, the composition of the invention,
or the contrast agent of the invention, for use in a diagnostic
method practised on the human or animal body. The diagnostic method
may for instance be a method for diagnosing a disease or condition
associated with hypoxia.
[0016] Detection of a condition is valuable, however methods of
treating the condition are also required. Therefore in yet another
aspect, the invention provides a method of evaluating the activity
of a pharmaceutical, which method comprises: [0017] (i)
administering to a subject the conjugate of the invention, the
composition of the invention, or the contrast agent of the
invention; [0018] (ii) detecting the conjugate in the subject by
MRI prior to administering the pharmaceutical to the subject;
[0019] (iii) administering the pharmaceutical to the subject;
[0020] (iv) detecting the conjugate in the subject by MRI after
administering the pharmaceutical to the subject; and [0021] (v)
evaluating changes in the MRI response of the conjugate before and
after administration of the pharmaceutical.
[0022] The pharmaceutical may for instance be a pharmaceutical for
the treatment, prevention or suppression of a disease or condition
associated with hypoxia.
[0023] Further provided is the conjugate of the invention, the
composition of the invention or the contrast agent of the
invention, for use in any one of the methods of the invention as
defined above.
BRIEF DESCRIPTION OF THE FIGURES
[0024] FIG. 1 shows a calibration curve used for determining the
amount of iron in the conjugate of the invention.
[0025] FIG. 2a shows cell mortality assays under hypoxic and
normoxic conditions in the presence and in absence of the conjugate
of the invention. FIG. 2b shows the relative cell mortality in
hypoxic conditions relative to normoxic conditions, in the presence
and in absence of the conjugate of the invention.
[0026] FIG. 3 shows a reaction scheme for the synthesis of a
conjugate of the invention.
[0027] FIG. 4 shows a calibration curve used for determining the
Fmoc modification of the conjugate, generated by measuring from
cleavage of Fmoc-Glycine.
[0028] FIG. 5 shows differential retention of the conjugate of the
invention following cellular growth in the presence of the
conjugate under hypoxic and normoxic conditions. Conditions: 1:
+conjugate/normoxic; 2: -conjugate/hypoxic; 3: +conjugate/hypoxic;
4: -conjugate/normoxic.
[0029] FIG. 6 shows 2D axial gradient echo images obtained on
agarose embedded cells, which had been subjected to, from left to
right: (1) normoxia+contrast; (2) normoxia+no contrast; (3)
hypoxia+contrast; (4) hypoxia+no contrast.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
[0030] The following substituent definitions apply with respect to
the compounds defined herein:
[0031] A C.sub.1-10 alkyl group is an unsubstituted or substituted,
straight or branched chain saturated hydrocarbon radical. Typically
it is C.sub.1-6 alkyl, for example methyl, ethyl, propyl, butyl,
pentyl or hexyl, or C.sub.1-4 alkyl, for example methyl, ethyl,
i-propyl, n-propyl, t-butyl, s-butyl or n-butyl. When an alkyl
group is substituted it typically bears one or more substituents
selected from substituted or unsubstituted C.sub.1-10 alkyl,
substituted or unsubstituted aryl (as defined herein), cyano,
amino, C.sub.1-10 alkylamino, di(C.sub.1-10)alkylamino, arylamino,
diarylamino, arylalkylamino, amido, acylamido, hydroxy, oxo, halo,
carboxy, ester, acyl, acyloxy, C.sub.1-10 alkoxy, aryloxy,
haloalkyl, sulfonic acid, sulfhydryl (i.e. thiol, --SH), C.sub.1-10
alkylthio, arylthio, sulfonyl, phosphoric acid, phosphate ester,
phosphonic acid and phosphonate ester. Examples of substituted
alkyl groups include haloalkyl, hydroxyalkyl, aminoalkyl,
alkoxyalkyl and alkaryl groups. The term alkaryl, as used herein,
pertains to a C.sub.1-10 alkyl group in which at least one hydrogen
atom has been replaced with an aryl group. Examples of such groups
include, but are not limited to, benzyl (phenylmethyl,
PhCH.sub.2--), benzhydryl (Ph.sub.2CH--), trityl (triphenylmethyl,
Ph.sub.3C--), phenethyl (phenylethyl, Ph-CH.sub.2CH.sub.2--),
styryl (Ph-CH.dbd.CH--), cinnamyl (Ph-CH.dbd.CH--CH.sub.2--).
Typically a substituted C.sub.1-10 alkyl group carries 1, 2 or 3
substituents, for instance 1 or 2.
[0032] A C.sub.2-10 alkenyl group is an unsubstituted or
substituted, straight or branched chain unsaturated hydrocarbon
radical having one or more, e.g. one or two, double bonds.
Typically it is C.sub.2-6 alkenyl, for example ethenyl, propenyl,
butenyl, pentenyl or hexenyl, or C.sub.2-4 alkenyl, for example
ethenyl, i-propenyl, n-propenyl, t-butenyl, s-butenyl or n-butenyl.
When an alkenyl group is substituted it typically bears one or more
substituents selected from substituted or unsubstituted C.sub.1-10
alkyl, substituted or unsubstituted aryl (as defined herein),
cyano, amino, C.sub.1-10 alkylamino, di(C.sub.1-10)alkylamino,
arylamino, diarylamino, arylalkylamino, amido, acylamido, hydroxy,
oxo, halo, carboxy, ester, acyl, acyloxy, C.sub.1-10 alkoxy,
aryloxy, haloalkyl, sulfonic acid, sulfhydryl (i.e. thiol, --SH),
C.sub.1-10 alkylthio, arylthio, sulfonyl, phosphoric acid,
phosphate ester, phosphonic acid and phosphonate ester. Examples of
substituted alkenyl groups include haloalkenyl, hydroxyalkenyl,
aminoalkenyl, alkoxyalkenyl and alkenaryl groups. The term
alkenaryl, as used herein, pertains to a C.sub.2-10 alkenyl group
in which at least one hydrogen atom has been replaced with an aryl
group. Examples of such groups include, but are not limited to,
styryl (PhCH.dbd.CH--), Ph.sub.2C.dbd.CH--, PhCH.dbd.C(Ph)-, and
cinnamyl (Ph-CH.dbd.CH--CH.sub.2--). Typically a substituted
C.sub.2-10 alkenyl group carries 1, 2 or 3 substituents, for
instance 1 or 2.
[0033] A C.sub.2-10 alkynyl group is an unsubstituted or
substituted, straight or branched chain unsaturated hydrocarbon
radical having one or more, e.g. one or two, triple bonds.
Typically it is C.sub.2-6 alkynyl, for example ethynyl, propynyl,
butynyl, pentynyl or hexynyl, or C.sub.2-4 alkynyl, for example
ethynyl, i-propynyl, n-propynyl, t-butynyl, s-butynyl or n-butynyl.
When an alkynyl group is substituted it typically bears one or more
substituents selected from substituted or unsubstituted C.sub.1-10
alkyl, substituted or unsubstituted aryl (as defined herein),
cyano, amino, C.sub.1-10 alkylamino, di(C.sub.1-10)alkylamino,
arylamino, diarylamino, arylalkylamino, amido, acylamido, hydroxy,
oxo, halo, carboxy, ester, acyl, acyloxy, C.sub.1-10 alkoxy,
aryloxy, haloalkyl, sulfonic acid, sulfhydryl (i.e. thiol, --SH),
C.sub.1-10 alkylthio, arylthio, sulfonyl, phosphoric acid,
phosphate ester, phosphonic acid and phosphonate ester. Examples of
substituted alkynyl groups include haloalkynyl, hydroxyalkynyl,
aminoalkynyl, alkoxyalkynyl and alkynaryl groups. The term
alkynaryl, as used herein, pertains to a C.sub.2-10 alkyl group in
which at least one hydrogen atom has been replaced with an aryl
group. Examples of such groups include, but are not limited to,
Ph-C.ident.C--, H--C.ident.C--CH(Ph)-, and
H--C.ident.C--CPh.sub.2-. Typically a substituted C.sub.2-10
alkynyl group carries 1, 2 or 3 substituents, for instance 1 or
2.
[0034] A C.sub.3-10 cycloalkyl group is an unsubstituted or
substituted alkyl group which is also a cyclyl group; that is, a
monovalent moiety obtained by removing a hydrogen atom from an
alicyclic ring atom of a carbocyclic ring of a carbocyclic
compound, which moiety has from 3 to 10 carbon atoms (unless
otherwise specified), including from 3 to 10 ring atoms. Thus, the
term "cycloalkyl" includes the sub-classes cycloalkyenyl and
cycloalkynyl. Examples of groups of C.sub.3-10 cycloalkyl groups
include C.sub.3-7 cycloalkyl. When a C.sub.3-10 cycloalkyl group is
substituted it typically bears one or more substituents selected
from C.sub.1-6 alkyl which is unsubstituted, aryl (as defined
herein), cyano, amino, C.sub.1-10 alkylamino,
di(C.sub.1-10)alkylamino, arylamino, diarylamino, arylalkylamino,
amido, acylamido, hydroxy, oxo, halo, carboxy, ester, acyl,
acyloxy, C.sub.1-20 alkoxy, aryloxy, haloalkyl, sulfonic acid,
sulfhydryl (i.e. thiol, --SH), C.sub.1-10 alkylthio, arylthio,
phosphoric acid, phosphate ester, phosphonic acid and phosphonate
ester and sulfonyl. Typically a substituted C.sub.3-10 cycloalkyl
group carries 1, 2 or 3 substituents, for instance 1 or 2.
[0035] Examples of C.sub.3-10 cycloalkyl groups include, but are
not limited to, those derived from saturated monocyclic hydrocarbon
compounds, which C.sub.3-10 cycloalkyl groups are unsubstituted or
substituted as defined above: cyclopropane (C.sub.3), cyclobutane
(C.sub.4), cyclopentane (C.sub.5), cyclohexane (C.sub.6),
cycloheptane (C.sub.7), methylcyclopropane (C.sub.4),
dimethylcyclopropane (C.sub.5), methylcyclobutane (C.sub.5),
dimethylcyclobutane (C.sub.6), methylcyclopentane (C.sub.6),
dimethylcyclopentane (C.sub.7), methylcyclohexane (C.sub.7),
dimethylcyclohexane (C.sub.8), menthane (C.sub.10);
[0036] unsaturated monocyclic hydrocarbon compounds: cyclopropene
(C.sub.3), cyclobutene (C.sub.4), cyclopentene (C.sub.5),
cyclohexene (C.sub.6), methylcyclopropene (C.sub.4),
dimethylcyclopropene (C.sub.5), methylcyclobutene (C.sub.5),
dimethylcyclobutene (C.sub.6), methylcyclopentene (C.sub.6),
dimethylcyclopentene (C.sub.7), methylcyclohexene (C.sub.7),
dimethylcyclohexene (C.sub.8);
[0037] saturated polycyclic hydrocarbon compounds: thujane
(C.sub.10), carane (C.sub.10), pinane (C.sub.10), bornane
(C.sub.10), norcarane (C.sub.7), norpinane (C.sub.7), norbornane
(C.sub.7), adamantane (C.sub.10), decalin (decahydronaphthalene)
(C.sub.10);
[0038] unsaturated polycyclic hydrocarbon compounds: camphene
(C.sub.10), limonene (C.sub.10), pinene (C.sub.10),
[0039] polycyclic hydrocarbon compounds having an aromatic ring:
indene (C.sub.9), indane (e.g., 2,3-dihydro-1H-indene) (C.sub.9),
tetraline (1,2,3,4-tetrahydronaphthalene) (C.sub.10).
[0040] A C.sub.3-10 heterocyclyl group is an unsubstituted or
substituted monovalent moiety obtained by removing a hydrogen atom
from a ring atom of a heterocyclic compound, which moiety has from
3 to 10 ring atoms (unless otherwise specified), of which from 1 to
5 are ring heteroatoms. Preferably, each ring has from 3 to 7 ring
atoms, of which from 1 to 4 are ring heteroatoms. When a C.sub.3-10
heterocyclyl group is substituted it typically bears one or more
substituents selected from C.sub.1-6 alkyl which is unsubstituted,
aryl (as defined herein), cyano, amino, C.sub.1-10 alkylamino,
di(C.sub.1-10)alkylamino, arylamino, diarylamino, arylalkylamino,
amido, acylamido, hydroxy, oxo, halo, carboxy, ester, acyl,
acyloxy, C.sub.1-20 alkoxy, aryloxy, haloalkyl, sulfonic acid,
sulfhydryl (i.e. thiol, --SH), C.sub.1-10 alkylthio, arylthio,
phosphoric acid, phosphate ester, phosphonic acid and phosphonate
ester and sulfonyl. Typically a substituted C.sub.3-10 heterocyclyl
group carries 1, 2 or 3 substituents, for instance 1 or 2.
[0041] Examples of groups of heterocyclyl groups include C.sub.5-10
heterocyclyl, C.sub.3-7 heterocyclyl, C.sub.5-7 heterocyclyl, and
C.sub.5-6 heterocyclyl.
[0042] Examples of (non-aromatic) monocyclic C.sub.3-10
heterocyclyl groups include, but are not limited to, those derived
from:
[0043] N.sub.1: aziridine (C.sub.3), azetidine (C.sub.4),
pyrrolidine (tetrahydropyrrole) (C.sub.5), pyrroline (e.g.,
3-pyrroline, 2,5-dihydropyrrole) (C.sub.5), 2H-pyrrole or
3H-pyrrole (isopyrrole, isoazole) (C.sub.5), piperidine (C.sub.6),
dihydropyridine (C.sub.6), tetrahydropyridine (C.sub.6), azepine
(C.sub.7);
[0044] O.sub.1: oxirane (C.sub.3), oxetane (C.sub.4), oxolane
(tetrahydrofuran) (C.sub.5), oxole (dihydrofuran) (C.sub.5), oxane
(tetrahydropyran) (C.sub.6), dihydropyran (C.sub.6), pyran
(C.sub.6), oxepin (C.sub.7);
[0045] S.sub.1: thiirane (C.sub.3), thietane (C.sub.4), thiolane
(tetrahydrothiophene) (C.sub.5), thiane (tetrahydrothiopyran)
(C.sub.6), thiepane (C.sub.7);
[0046] O.sub.2: dioxolane (C.sub.5), dioxane (C.sub.6), and
dioxepane (C.sub.7);
[0047] O.sub.3: trioxane (C.sub.6);
[0048] N.sub.2: imidazolidine (C.sub.5), pyrazolidine (diazolidine)
(C.sub.5), imidazoline (C.sub.5), pyrazoline (dihydropyrazole)
(C.sub.5), piperazine (C.sub.6);
[0049] N.sub.1O.sub.1: tetrahydrooxazole (C.sub.5), dihydrooxazole
(C.sub.5), tetrahydroisoxazole (C.sub.5), dihydroisoxazole
(C.sub.5), morpholine (C.sub.6), tetrahydrooxazine (C.sub.6),
dihydrooxazine (C.sub.6), oxazine (C.sub.6);
[0050] N.sub.1S.sub.1: thiazoline (C.sub.5), thiazolidine
(C.sub.5), thiomorpholine (C.sub.6);
[0051] N.sub.2O.sub.1: oxadiazine (C.sub.6);
[0052] O.sub.1S.sub.1: oxathiole (C.sub.5) and oxathiane (thioxane)
(C.sub.6); and,
[0053] N.sub.1O.sub.1S.sub.1: oxathiazine (C.sub.6).
[0054] Examples of substituted (non-aromatic) monocyclic
heterocyclyl groups include those derived from saccharides, in
cyclic form, for example, furanoses (C.sub.5), such as
arabinofuranose, lyxofuranose, ribofuranose, and xylofuranse, and
pyranoses (C.sub.6), such as allopyranose, altropyranose,
glucopyranose, mannopyranose, gulopyranose, idopyranose,
galactopyranose, and talopyranose.
[0055] Examples of C.sub.3-10 heterocyclyl groups which are also
aryl groups are described below as heteroaryl groups.
[0056] An aryl group is a substituted or unsubstituted, monocyclic
or fused polycyclic aromatic group which typically contains from 6
to 14 carbon atoms, preferably from 6 to 10 carbon atoms, in the
ring portion. Examples include phenyl (i.e. monocyclic), naphthyl,
indenyl and indanyl (i.e. fused bicyclic), anthracenyl (i.e. fused
tricyclic), and pyrenyl (i.e. fused tetracyclic) groups. An aryl
group is unsubstituted or substituted. When an aryl group as
defined above is substituted it typically bears one or more
substituents selected from C.sub.1-C.sub.6 alkyl which is
unsubstituted (to form an aralkyl group), aryl which is
unsubstituted, cyano, amino, C.sub.1-10 alkylamino,
di(C.sub.1-10)alkylamino, arylamino, diarylamino, arylalkylamino,
amido, acylamido, hydroxy, halo, carboxy, ester, acyl, acyloxy,
C.sub.1-20 alkoxy, aryloxy, haloalkyl, sulfhydryl (i.e. thiol,
--SH), C.sub.1-10 alkylthio, arylthio, sulfonic acid, phosphoric
acid, phosphate ester, phosphonic acid and phosphonate ester and
sulfonyl. Typically it carries 0, 1, 2 or 3 substituents. A
substituted aryl group may be substituted in two positions with a
single C.sub.1-6 alkylene group, or with a bidentate group
represented by the formula --X--C.sub.1-6 alkylene, or
--X--C.sub.1-6 alkylene-X--, wherein X is selected from 0, S and
NR, and wherein R is H, aryl or C.sub.1-6 alkyl. Thus a substituted
aryl group may be an aryl group fused with a cycloalkyl group or
with a heterocyclyl group. The term aralkyl as used herein,
pertains to an aryl group in which at least one hydrogen atom
(e.g., 1, 2, 3) has been substituted with a C.sub.1-6 alkyl group.
Examples of such groups include, but are not limited to, tolyl
(from toluene), xylyl (from xylene), mesityl (from mesitylene), and
cumenyl (or cumyl, from cumene), and duryl (from durene).
[0057] As used herein, a heteroaryl group is a substituted or
unsubstituted monocyclic or fused polycyclic (e.g. bicyclic or
tricyclic) aromatic group which typically contains from 5 to 14
atoms in the ring portion including at least one heteroatom, for
example 1, 2 or 3 heteroatoms, selected from O, S, N, P, Se and Si,
more typically from O, S and N. Examples include pyridyl,
pyrazinyl, pyrimidinyl, pyridazinyl, furanyl, thienyl,
pyrazolidinyl, pyrrolyl, oxadiazolyl, isoxazolyl, thiadiazolyl,
thiazolyl, imidazolyl, triazolyl, pyrazolyl, oxazolyl,
isothiazolyl, benzofuranyl, isobenzofuranyl, benzothiophenyl,
indolyl, indazolyl, carbazolyl, acridinyl, purinyl, cinnolinyl,
quinoxalinyl, naphthyridinyl, benzimidazolyl, benzoxazolyl,
quinolinyl, quinazolinyl and isoquinolinyl. A heteroaryl group is
often a 5- or 6-membered ring. However, as used herein, references
to a heteroaryl group also include fused polycyclic ring systems,
including for instance fused bicyclic systems in which a heteroaryl
group is fused to an aryl group. When the heteroaryl group is such
a fused heteroaryl group, preferred examples are fused ring systems
wherein a 5- to 6-membered heteroaryl group is fused to a phenyl
group. Examples of such fused ring systems are benzofuranyl,
isobenzofuranyl, benzothiophenyl, indolyl, indazolyl,
benzimidazolyl, benzoxazolyl, quinolinyl, quinazolinyl and
isoquinolinyl moieties.
[0058] A heteroaryl group may be unsubstituted or substituted, for
instance, as specified above for aryl. Typically it carries 0, 1, 2
or 3 substituents.
[0059] A C.sub.1-10 alkylene group is an unsubstituted or
substituted bidentate moiety obtained by removing two hydrogen
atoms, either both from the same carbon atom, or one from each of
two different carbon atoms, of a hydrocarbon compound having from 1
to 10 carbon atoms (unless otherwise specified), which may be
aliphatic or alicyclic, and which may be saturated, partially
unsaturated, or fully unsaturated. Thus, the term "alkylene"
includes the sub-classes alkenylene, alkynylene, cycloalkylene,
etc., discussed below. Usually, however, it is a saturated
aliphatic (non-cyclic) group. Typically it is C.sub.1-6 alkylene,
or C.sub.1-4 alkylene, for example methylene, ethylene,
i-propylene, n-propylene, t-butylene, s-butylene or n-butylene. It
may for instance be C.sub.2-4 alkylene. Or, for instance, it may be
C.sub.2-3 alkylene, for example ethylene, n-propylene or
i-propylene. (Although usually, herein, a C.sub.2-3 alkylene refers
to ethylene or n-proylene.) It may also be pentylene, hexylene,
heptylene, octylene and the various branched chain isomers thereof.
An alkylene group may be unsubstituted or substituted, for
instance, as specified above for alkyl. Typically a substituted
alkylene group carries 1, 2 or 3 substituents, for instance 1 or
2.
[0060] In this context, the prefixes (e.g., C.sub.1-4, C.sub.1-7,
C.sub.1-10, C.sub.2-7, C.sub.3-7, etc.) denote the number of carbon
atoms, or range of number of carbon atoms. For example, the term
"C.sub.1-4 alkylene," as used herein, pertains to an alkylene group
having from 1 to 4 carbon atoms. Examples of groups of alkylene
groups include C.sub.1-4 alkylene ("lower alkylene"), C.sub.1-7
alkylene, and C.sub.1-10 alkylene.
[0061] Examples of linear saturated C.sub.1-7 alkylene groups
include, but are not limited to, --(CH.sub.2).sub.n-- where n is an
integer from 1 to 7, for example, --CH.sub.2-- (methylene),
--CH.sub.2CH.sub.2-- (ethylene), --CH.sub.2CH.sub.2CH.sub.2--
(propylene), and --CH.sub.2CH.sub.2CH.sub.2CH.sub.2--
(butylene).
[0062] Examples of branched saturated C.sub.1-7 alkylene groups
include, but are not limited to, --CH(CH.sub.3)--,
--CH(CH.sub.3)CH.sub.2--, --CH(CH.sub.3)CH.sub.2CH.sub.2--,
--CH(CH.sub.3)CH.sub.2CH.sub.2CH.sub.2--,
--CH.sub.2CH(CH.sub.3)CH.sub.2--,
--CH.sub.2CH(CH.sub.3)CH.sub.2CH.sub.2--, --CH(CH.sub.2CH.sub.3)--,
--CH(CH.sub.2CH.sub.3)CH.sub.2--, and
--CH.sub.2CH(CH.sub.2CH.sub.3)CH.sub.2--.
[0063] Examples of linear partially unsaturated C.sub.1-7 alkylene
groups include, but is not limited to, --CH.dbd.CH-- (vinylene),
--CH.dbd.CH--CH.sub.2--, --CH.sub.2--CH.dbd.CH.sub.2--,
--CH.dbd.CH--CH.sub.2--CH.sub.2--,
--CH.dbd.CH--CH.sub.2--CH.sub.2--CH.sub.2--,
--CH.dbd.CH--CH.dbd.CH--, --CH.dbd.CH--CH.dbd.CH--CH.sub.2--,
--CH.dbd.CH--CH.dbd.CH--CH.sub.2--CH.sub.2--,
--CH.dbd.CH--CH.sub.2--CH.dbd.CH--, and
--CH.dbd.CH--CH.sub.2--CH.sub.2--CH.dbd.CH--.
[0064] Examples of branched partially unsaturated C.sub.1-7
alkylene groups include, but is not limited to,
--C(CH.sub.3).dbd.CH--, --C(CH.sub.3).dbd.CH--CH.sub.2--, and
--CH.dbd.CH--CH(CH.sub.3)--.
[0065] Examples of alicyclic saturated C.sub.1-7 alkylene groups
include, but are not limited to, cyclopentylene (e.g.,
cyclopent-1,3-ylene), and cyclohexylene (e.g.,
cyclohex-1,4-ylene).
[0066] Examples of alicyclic partially unsaturated C.sub.1-7
alkylene groups include, but are not limited to, cyclopentenylene
(e.g., 4-cyclopenten-1,3-ylene), cyclohexenylene (e.g.,
2-cyclohexen-1,4-ylene; 3-cyclohexen-1,2-ylene;
2,5-cyclohexadien-1,4-ylene).
[0067] C.sub.1-10 alkylene and C.sub.1-10 alkyl groups as defined
herein are either uninterrupted or interrupted by one or more
heteroatoms or heterogroups, such as S, O or N(R'') wherein R'' is
H, C.sub.1-6 alkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, aryl
(typically phenyl), or heteroaryl, or by one or more arylene or
heteroarylene (typically arylene, more typically phenylene) groups,
or by one or more --C(O)--, --C(O)O-- or --C(O)N(R'')-- groups. The
phrase "optionally interrupted" as used herein thus refers to a
C.sub.1-10 alkyl group or an alkylene group, as defined above,
which is uninterrupted or which is interrupted between adjacent
carbon atoms by a heteroatom such as oxygen or sulfur, by a
heterogroup such as N(R'') wherein R'' is H, aryl, heteroaryl or
C.sub.1-6 alkyl, or by an arylene or heteroarylene (typically
arylene, more typically phenylene) group, or by a --C(O)--,
--C(O)O-- or --C(O)N(R'')-- group, again wherein R'' is H, aryl or
C.sub.1-6 alkyl.
[0068] For instance, a C.sub.1-10 alkyl group such as n-butyl may
be interrupted by the heterogroup N(R'') as follows:
--CH.sub.2N(R'')CH.sub.2CH.sub.2CH.sub.3,
--CH.sub.2CH.sub.2N(R'')CH.sub.2CH.sub.3, or
--CH.sub.2CH.sub.2CH.sub.2N(R'')CH.sub.3. Similarly, an alkylene
group such as n-butylene may be interrupted by the heterogroup
N(R'') as follows: --CH.sub.2N(R'')CH.sub.2CH.sub.2CH.sub.2--,
--CH.sub.2CH.sub.2N(R'')CH.sub.2CH.sub.2--, or
--CH.sub.2CH.sub.2CH.sub.2N(R'')CH.sub.2--. Typically an
interrupted group, for instance an interrupted C.sub.1-10 alkylene
or C.sub.1-10 alkyl group, is interrupted by 1, 2 or 3 heteroatoms
or heterogroups or by 1, 2 or 3 arylene (typically phenylene)
groups. More typically, an interrupted group, for instance an
interrupted C.sub.1-10 alkylene or C.sub.1-10 alkyl group, is
interrupted by 1 or 2 heteroatoms or heterogroups or by 1 or 2
arylene (typically phenylene) groups. For instance, a C.sub.1-20
alkyl group such as n-butyl may be interrupted by 2 heterogroups
N(R'') as follows:
--CH.sub.2N(R'')CH.sub.2N(R'')CH.sub.2CH.sub.3.
[0069] An arylene group is an unsubstituted or substituted
monocyclic or fused polycyclic bidentate moiety obtained by
removing two hydrogen atoms, one from each of two different
aromatic ring atoms of an aromatic compound, which moiety has from
5 to 14 ring atoms (unless otherwise specified). Typically, each
ring has from 5 to 7 or from 5 to 6 ring atoms. An arylene group
may be unsubstituted or substituted, for instance, as specified
above for aryl.
[0070] In this context, the prefixes (e.g., C.sub.5-20, C.sub.6-20,
C.sub.5-14, C.sub.5-7, C.sub.5-6, etc.) denote the number of ring
atoms, or range of number of ring atoms, whether carbon atoms or
heteroatoms. For example, the term "C.sub.5-6 arylene," as used
herein, pertains to an arylene group having 5 or 6 ring atoms.
Examples of groups of arylene groups include C.sub.5-20 arylene,
C.sub.6-20 arylene, C.sub.5-14 arylene, C.sub.6-14 arylene,
C.sub.6-10 arylene, C.sub.5-12 arylene, C.sub.5-10 arylene,
C.sub.5-7 arylene, C.sub.5-6 arylene, C.sub.5 arylene, and C.sub.6
arylene.
[0071] The ring atoms may be all carbon atoms, as in "carboarylene
groups" (e.g., C.sub.6-20 carboarylene, C.sub.6-14 carboarylene or
C.sub.6-10 carboarylene).
[0072] Examples of C.sub.6-20 arylene groups which do not have ring
heteroatoms (i.e., C.sub.6-20 carboarylene groups) include, but are
not limited to, those derived from the compounds discussed above in
regard to aryl groups, e.g. phenylene, and also include those
derived from aryl groups which are bonded together, e.g.
phenylene-phenylene (diphenylene) and phenylene-phenylene-phenylene
(triphenylene). Alternatively, the ring atoms may include one or
more heteroatoms, as in "heteroarylene groups" (e.g., C.sub.5-14
heteroarylene). Examples of C.sub.5-14 heteroarylene groups
include, but are not limited to, those derived from the compounds
discussed above in regard to heteroaryl groups.
[0073] As used herein the term oxo represents a group of formula:
.dbd.O
[0074] As used herein the term acyl represents a group of formula:
--C(.dbd.O)R, wherein R is an acyl substituent, for example, a
substituted or unsubstituted C.sub.1-20 alkyl group, a C.sub.1-20
perfluoroalkyl group, a substituted or unsubstituted C.sub.3-10
cycloalkyl group, a substituted or unsubstituted C.sub.3-10
heterocyclyl group, a substituted or unsubstituted aryl group, a
perfluoroaryl group, or a substituted or unsubstituted heteroaryl
group. Examples of acyl groups include, but are not limited to,
--C(.dbd.O)CH.sub.3 (acetyl), --C(.dbd.O)CH.sub.2CH.sub.3
(propionyl), --C(.dbd.O)C(CH.sub.3).sub.3 (t-butyryl), and
--C(.dbd.O)Ph (benzoyl, phenone).
[0075] As used herein the term acyloxy (or reverse ester)
represents a group of formula: --OC(.dbd.O)R, wherein R is an
acyloxy substituent, for example, substituted or unsubstituted
C.sub.1-10 alkyl group, a substituted or unsubstituted C.sub.3-10
heterocyclyl group, or a substituted or unsubstituted aryl group,
typically a C.sub.1-6 alkyl group. Examples of acyloxy groups
include, but are not limited to, --OC(.dbd.O)CH.sub.3 (acetoxy),
--OC(.dbd.O)CH.sub.2CH.sub.3, --OC(.dbd.O)C(CH.sub.3).sub.3,
--OC(.dbd.O)Ph, and --OC(.dbd.O)CH.sub.2Ph.
[0076] As used herein the term ester (or carboxylate, carboxylic
acid ester or oxycarbonyl) represents a group of formula:
--C(.dbd.O)OR, wherein R is an ester substituent, for example, a
substituted or unsubstituted C.sub.1-10 alkyl group, a substituted
or unsubstituted C.sub.3-20 heterocyclyl group, or a substituted or
unsubstituted aryl group (typically a phenyl group). Examples of
ester groups include, but are not limited to, --C(.dbd.O)OCH.sub.3,
--C(.dbd.O)OCH.sub.2CH.sub.3, --C(.dbd.O)OC(CH.sub.3).sub.3, and
--C(.dbd.O)OPh.
[0077] As used herein the term amino represents a group of formula
--NH.sub.2. The term C.sub.1-10 alkylamino represents a group of
formula --NHR' wherein R' is a C.sub.1-10 alkyl group, preferably a
C.sub.1-6 alkyl group, as defined previously. The term
di(C.sub.1-10)alkylamino represents a group of formula --NR'R''
wherein R' and R'' are the same or different and represent
C.sub.1-10 alkyl groups, preferably C.sub.1-6 alkyl groups, as
defined previously. The term arylamino represents a group of
formula --NHR' wherein R' is an aryl group, preferably a phenyl
group, as defined previously. The term diarylamino represents a
group of formula --NR'R'' wherein R' and R'' are the same or
different and represent aryl groups, preferably phenyl groups, as
defined previously. The term arylalkylamino represents a group of
formula --NR'R'' wherein R' is a C.sub.1-10 alkyl group, preferably
a C.sub.1-6 alkyl group, and R'' is an aryl group, preferably a
phenyl group.
[0078] A halo group is chlorine, fluorine, bromine or iodine (a
chloro group, a fluoro group, a bromo group or an iodo group). It
is typically chlorine, fluorine or bromine.
[0079] As used herein the term amido represents a group of formula:
--C(.dbd.O)NR'R'', wherein R' and R'' are independently selected
from H, C.sub.1-10 alkyl and aryl. Examples of amido groups
include, but are not limited to, --C(.dbd.O)NH.sub.2,
--C(.dbd.O)NHCH.sub.3, --C(.dbd.O)N(CH.sub.3).sub.2,
--C(.dbd.O)NHCH.sub.2CH.sub.3, and
--C(.dbd.O)N(CH.sub.2CH.sub.3).sub.2, as well as amido groups in
which R' and R'', together with the nitrogen atom to which they are
attached, form a heterocyclic structure as in, for example,
piperidinocarbonyl, morpholinocarbonyl, thiomorpholinocarbonyl, and
piperazinocarbonyl.
[0080] As used herein the term acylamido represents a group of
formula: --NR.sup.1C(.dbd.O)R.sup.2, wherein R.sup.1 is an amide
substituent, for example, hydrogen, a C.sub.1-10 alkyl group, a
C.sub.3-20 heterocyclyl group, an aryl group, preferably hydrogen
or a C.sub.1-10 alkyl group, and R.sup.2 is an acyl substituent,
for example, a C.sub.1-10 alkyl group, a C.sub.3-20 heterocyclyl
group, or an aryl group. Preferably R.sup.1 is hydrogen and R.sup.2
is a C.sub.1-10 alkyl group. Examples of acylamide groups include,
but are not limited to, --NHC(.dbd.O)CH.sub.3,
--NHC(.dbd.O)CH.sub.2CH.sub.3, --NHC(.dbd.O)Ph,
--NHC(.dbd.O)C.sub.15H.sub.31 and --NHC(.dbd.O)C.sub.9H.sub.19.
Thus, a substituted C.sub.1-10 alkyl group may comprise an
acylamido substituent defined by the formula
--NHC(.dbd.O)--C.sub.1-10 alkyl, such as
--NHC(.dbd.O)C.sub.5H.sub.11 or --NHC(.dbd.O)C.sub.9H.sub.19.
R.sup.1 and R.sup.2 may together form a cyclic structure, as in,
for example, succinimidyl, maleimidyl, and phthalimidyl:
##STR00001##
[0081] A C.sub.1-10 alkylthio group is a said C.sub.1-10 alkyl
group, preferably a C.sub.1-6 alkyl group, attached to a thio
group. An arylthio group is an aryl group, preferably a phenyl
group, attached to a thio group.
[0082] A C.sub.1-10 alkoxy group is a said substituted or
unsubstituted C.sub.1-10 alkyl group attached to an oxygen atom. A
C.sub.1-6 alkoxy group is a said substituted or unsubstituted
C.sub.1-6 alkyl group attached to an oxygen atom. A C.sub.1-4
alkoxy group is a substituted or unsubstituted C.sub.1-4 alkyl
group attached to an oxygen atom. Said C.sub.1-10, C.sub.1-6 and
C.sub.1-4 alkyl groups are optionally interrupted as defined
herein. Examples of C.sub.1-4 alkoxy groups include, --OMe
(methoxy), --OEt (ethoxy), --O(nPr) (n-propoxy), --O(iPr)
(isopropoxy), --O(nBu) (n-butoxy), --O(sBu) (sec-butoxy), --O(iBu)
(isobutoxy), and --O(tBu) (tert-butoxy). Further examples of
C.sub.1-20 alkoxy groups are --O(Adamantyl),
--O--CH.sub.2-Adamantyl and --O--CH.sub.2--CH.sub.2-Adamantyl. An
aryloxy group is a substituted or unsubstituted aryl group, as
defined herein, attached to an oxygen atom. An example of an
aryloxy group is --OPh (phenoxy).
[0083] Unless otherwise specified, included in the above are the
well known ionic, salt, solvate, and protected forms of these
substituents. For example, a reference to carboxylic acid, carboxy
or carboxyl group (--COOH) also includes the anionic (carboxylate)
form (--COO), a salt or solvate thereof, as well as conventional
protected forms. Similarly, a reference to an amino group includes
the protonated form (--N.sup.+HR.sup.1R.sup.2), a salt or solvate
of the amino group, for example, a hydrochloride salt, as well as
conventional protected forms of an amino group. Similarly, a
reference to a hydroxy or hydroxyl group (--OH) also includes the
anionic form (--O.sup.-), a salt or solvate thereof, as well as
conventional protected forms.
[0084] Certain compounds may exist in one or more particular
geometric, optical, enantiomeric, diasteriomeric, epimeric,
atropic, stereoisomeric, tautomeric, conformational, or anomeric
forms, including but not limited to, cis- and trans-forms; E- and
Z-forms; c-, t-, and r-forms; endo- and exo-forms; R-, S-, and
meso-forms; D- and L-forms; d- and 1-forms; (+) and (-) forms;
keto-, enol-, and enolate-forms; syn- and anti-forms; synclinal-
and anticlinal-forms; .alpha.- and .beta.-forms; axial and
equatorial forms; boat-, chair-, twist-, envelope-, and
halfchair-forms; and combinations thereof, hereinafter collectively
referred to as "isomers" (or "isomeric forms").
[0085] Note that, except as discussed below for tautomeric forms,
specifically excluded from the term "isomers," as used herein, are
structural (or constitutional) isomers (i.e., isomers which differ
in the connections between atoms rather than merely by the position
of atoms in space). For example, a reference to a methoxy group,
--OCH.sub.3, is not to be construed as a reference to its
structural isomer, a hydroxymethyl group, --CH.sub.2OH. Similarly,
a reference to ortho-chlorophenyl is not to be construed as a
reference to its structural isomer, meta-chlorophenyl. However, a
reference to a class of structures may well include structurally
isomeric forms falling within that class (e.g., C.sub.1-7alkyl
includes n-propyl and iso-propyl; butyl includes n-, iso-, sec-,
and tert-butyl; methoxyphenyl includes ortho-, meta-, and
para-methoxyphenyl).
[0086] The above exclusion does not pertain to tautomeric forms,
for example, keto, enol, and enolate forms, as in, for example, the
following tautomeric pairs: keto/enol (illustrated below),
imine/enamine, amide/imino alcohol, amidine/amidine, nitroso/oxime,
thioketone/enethiol, N-nitroso/hyroxyazo, and nitro/aci-nitro.
##STR00002##
[0087] Unless otherwise specified, a reference to a particular
compound includes all such isomeric forms, including (wholly or
partially) racemic and other mixtures thereof. Methods for the
preparation (e.g., asymmetric synthesis) and separation (e.g.,
fractional crystallisation and chromatographic means) of such
isomeric forms are either known in the art or are readily obtained
by adapting known methods, in a known manner.
[0088] Unless otherwise specified, a reference to a particular
compound or complex also includes ionic, salt, solvated and
protected forms.
Conjugates
[0089] The invention provides a conjugate which comprises an MRI
contrast agent component comprising a nanoparticle, which
nanoparticle comprises a metal or a metal compound, and a hypoxia
targeting moiety which is conjugated to the MRI contrast agent
component. In some cases, only one hypoxia targeting moiety is
conjugated to the MRI contrast agent component. Often, however,
more than one hypoxia targeting moiety is conjugated to the MRI
contrast component. Thus, the conjugate may comprise a plurality of
hypoxia targeting moieties conjugated to the MRI contrast agent
component. An advantage of using a nanoparticle as the contrast
agent component is that many hypoxia targeting moieties may be
conjugated to the contrast agent component in a single conjugate,
which increases the conjugate's affinity for hypoxic tissue.
[0090] The term "nanoparticle", as used herein, means a microscopic
particle whose size is typically measured in nanometres. A
nanoparticle typically has a particle size of from 0.5 nm to 1000
nm. A nanoparticle often has a particle size of from 1 nm to 200
nm, more typically from 1 nm to 100 nm, for instance from 10 nm to
80 nm. A nanoparticle may be spherical or non-spherical.
Non-spherical nanoparticles may for instance be plate-shaped,
needle-shaped or tubular. The term "particle size" as used herein
means the diameter of the particle if the particle is spherical,
or, if the particle is non-spherical, the volume-based particle
size. The volume-based particle size is the diameter of a sphere
that has the same volume as the non-spherical particle in
question.
[0091] As used herein, the term "conjugated" refers to the or each
hypoxia targeting moiety being bound either directly or indirectly
to the MRI contrast component. For example, the or each hypoxia
targeting moiety may be covalently or non-covalently bonded to the
MRI contrast component. The moiety may be covalently or
non-covalently bonded directly to the MRI contrast component.
Alternatively, the hypoxia targeting moiety may be bonded via a
linker group or a spacer group to the MRI contrast component.
Typically, the or each hypoxia targeting moiety is covalently
bonded to the MRI contrast component via a linker group. Thus,
usually, the or each hypoxia targeting moiety is conjugated to the
MRI contrast component via a linker group. Often the or each
hypoxia targeting moiety is covalently bonded to the MRI contrast
component via a linker group.
[0092] The term "hypoxia targeting moiety", as used herein, relates
to a chemical moiety which is capable of targeting regions of
hypoxia in vivo or in vitro. The term "chemical moiety" as used
herein takes its usual meaning in the art, and relates to any
chemical group or compound. A hypoxia targeting moiety targets
regions of hypoxia by accumulating in these regions. For example,
the moiety may be sequestered in hypoxic regions, or may chemically
or physically bind (either permanently or transiently) to groups
which accumulate or have higher abundance in hypoxic regions.
Hypoxia targeting moieties may target physiological markers of
hypoxia such as pH, ionic conductivity, or temperature which may be
associated with the condition of hypoxia, and thus target regions
of hypoxia. Alternatively, hypoxia targeting moieties may be
altered, destroyed or otherwise chemically changed by chemicals
present in non-hypoxic regions, and thus accumulate in hypoxic
regions. For example, a hypoxia targeting moiety may be oxidised by
oxygen in normoxic regions.
[0093] Typically, the or each hypoxia targeting moiety comprises a
heteroaryl group, which heteroaryl group is substituted by a nitro
group, and is otherwise unsubstituted or substituted. For example,
the heteroaryl group may be an imidazolyl group, a pyrimidine
group, an oxazine group, a thiazine group, a triazole group, or a
triazine group, provided that said heteroaryl group is substituted
with a nitro group. The heteroaryl group may be otherwise
unsubstituted or may be further substituted by 1, 2 or 3
substituents as described hereinbefore for aryl and heteroaryl
groups. Thus, the or each hypoxia targeting moiety may comprise an
imidazolyl group, which imidazolyl group is substituted by a nitro
group, and is otherwise unsubstituted or substituted (as defined
hereinbefore for aryl and heteroaryl groups).
[0094] Usually, the or each hypoxia targeting moiety is an
unsubstituted or substituted 2-nitroimidazolyl group. The
unsubstituted or substituted 2-nitroimidazolyl group may for
instance be a group of formula (I):
##STR00003##
[0095] In formula (I), one of R.sup.1, R.sup.2 and R.sup.3 is a
bond attaching the hypoxia targeting moiety to the MRI contrast
agent component or to a linker group which is bonded to the MRI
contrast agent component; and the other two of R.sup.1, R.sup.2 and
R.sup.3, which are the same or different, are independently
selected from H, OH, halo, unsubstituted or substituted C.sub.1-10
alkyl, unsubstituted or substituted C.sub.2-10 alkenyl,
unsubstituted or substituted C.sub.2-10 alkynyl, unsubstituted or
substituted aryl, unsubstituted or substituted heteroaryl,
unsubstituted or substituted C.sub.3-10 cycloalkyl, unsubstituted
or substituted C.sub.3-10 heterocyclyl, unsubstituted or
substituted C.sub.1-10 alkoxy, unsubstituted or substituted
aryloxy, --N(R.sup.4).sub.2, --NO.sub.2, SR.sup.4,
--SO.sub.2R.sup.4, --CN, --C(O)R.sup.4, --OC(O)R.sup.4,
--C(O)OR.sup.4, --C(O)N(R.sup.4).sub.2, --NR.sup.4C(.dbd.O)R.sup.4
and --OP(O)(OR.sup.4).sub.2, provided that, when the other two of
R.sup.1, R.sup.2 and R.sup.3 are at adjacent positions on the
imidazolyl ring, said other two of R.sup.1, R.sup.2 and R.sup.3 may
together form an unsubstituted or substituted C.sub.2-4 alkylene
group; and the or each R.sup.4 is independently selected from H,
unsubstituted or substituted C.sub.1-6 alkyl, unsubstituted or
substituted C.sub.2-6 alkenyl, unsubstituted or substituted
C.sub.2-6 alkynyl, unsubstituted or substituted aryl, unsubstituted
or substituted heteroaryl, unsubstituted or substituted C.sub.3-10
cycloalkyl, and unsubstituted or substituted C.sub.3-10
heterocyclyl.
[0096] Often, one of R.sup.1, R.sup.2 and R.sup.3 is a bond
attaching the hypoxia targeting moiety to a linker group which is
bonded to the MRI contrast agent component, and the other two of
R.sup.1, R.sup.2 and R.sup.3 are as defined above. For instance,
R.sup.3 may be a bond attaching the hypoxia targeting moiety to a
linker group which is bonded to the MRI contrast agent component,
and R.sup.1 and R.sup.2 which may be the same or different, are
independently selected from H, OH, halo, unsubstituted or
substituted C.sub.1-10 alkyl, unsubstituted or substituted
C.sub.2-10 alkenyl, unsubstituted or substituted C.sub.2-10
alkynyl, unsubstituted or substituted aryl, unsubstituted or
substituted heteroaryl, unsubstituted or substituted C.sub.3-10
cycloalkyl, unsubstituted or substituted C.sub.3-10 heterocyclyl,
unsubstituted or substituted C.sub.1-10 alkoxy, unsubstituted or
substituted aryloxy, --N(R.sup.4).sub.2, --NO.sub.2, SR.sup.4,
--SO.sub.2R.sup.4, --CN, --C(O)R.sup.4, --OC(O)R.sup.4,
--C(O)OR.sup.4, --C(O)N(R.sup.4).sub.2, --NR.sup.4C(.dbd.O)R.sup.4
and --OP(O)(OR.sup.4).sub.2, provided that R.sup.1 and R.sup.2 may
together form an unsubstituted or substituted C.sub.2-4 alkylene
group. The or each R.sup.4 may be as defined above.
[0097] Usually, for instance, R.sup.3 is a bond attaching the
hypoxia targeting moiety to a linker group which is bonded to the
MRI contrast agent, and R.sup.1 and R.sup.2 are independently
selected from H, OH, halo, NH.sub.2, and unsubstituted or
substituted C.sub.1-3 alkyl. For example, R.sup.1 and R.sup.2 may
both be H.
[0098] As used herein, the term "linker" relates to a chemical
moiety which serves to bind the hypoxia targeting moiety to the MRI
contrast component and/or to maintain distance between the hypoxia
targeting moiety and the MRI contrast component. The terms "linker"
and "linker group" may be used interchangeably. When the
nanoparticle of the MRI contrast component comprises a coating, the
linker or linker group may bind the hypoxia targeting moiety to the
coating. Alternatively, the linker or linker group may bind to an
uncoated region of the MRI contrast component. Usually, however, it
binds to the coating, when a coating is present.
[0099] Typically, the linker group of the conjugate of the
invention is a group of formula (II):
##STR00004## [0100] In formula (II), *is the point of attachment of
the group of formula (II) to the hypoxia targeting moiety; [0101] X
is NR.sup.5 or O; [0102] L.sup.1 is unsubstituted or substituted
C.sub.1-10 alkylene which C.sub.1-10 alkylene is optionally
interrupted by N(R.sup.5), O, S, C(O), C(O)N(R.sup.5),
N(R.sup.5)C(O), OC(O), C(O)O, arylene or heteroarylene; [0103] n is
0 or an integer of from 1 to 3; [0104] the or each Y is
independently selected from S, N(R.sup.5), O, C(O), C(O)N(R.sup.5),
N(R.sup.5)C(O), OC(O), C(O)O, C(R.sup.5).sub.2, arylene and
heteroarylene; [0105] L.sup.2 is unsubstituted or substituted
C.sub.1-10 alkylene which C.sub.1-10 alkylene is optionally
interrupted by N(R.sup.5), O, S, C(O), C(O)N(R.sup.5),
N(R.sup.5)C(O), OC(O), C(O)O, arylene or heteroarylene; [0106] Z is
a bond attaching the group of formula (II) to the MRI contrast
agent component; and [0107] the or each R.sup.5 is independently
selected from H, unsubstituted or substituted C.sub.1-6 alkyl,
unsubstituted or substituted C.sub.2-6 alkenyl, unsubstituted or
substituted C.sub.2-6 alkynyl, unsubstituted or substituted aryl,
and unsubstituted or substituted heteroaryl. Typically, n is 0.
[0108] Usually, X is NH; L.sup.1 is CH.sub.2; n is 0 or 1; Y is S;
and L.sup.2 is unsubstituted C.sub.1-10 alkylene which is
optionally interrupted by N(R.sup.5), O, S, C(O), C(O)N(R.sup.5),
N(R.sup.5)C(O), OC(O) or C(O)O, wherein R.sup.5 is as defined
above. Typically, n is 0.
[0109] Usually, Z is a bond attaching the group of formula (II) to
a functional group in the MRI contrast agent component. Any
suitable functional group can be employed in the MRI contrast agent
component. Often, however, the functional group is an amine group,
an alcohol, or a carboxyl group.
[0110] An amine group of the contrast agent component is usually an
amino group (H(R)N--*, wherein R is H), a C.sub.1-10 alkylamino
group as defined hereinabove (i.e. a group H(R)N--*, wherein R is a
C.sub.1-10 alkyl group), or an arylamino group as defined
hereinabove (i.e. a group H(R)N--*, wherein R is an aryl group),
wherein * is the point of attachment of the amine group to the
contrast agent component. When the group of formula (II) is bonded
to the amine group by Z, a hydrogen of the amine group is typically
replaced by Z. Therefore, when the group of formula (II) is bonded
to the contrast agent component by Z, the amine group of the
contrast agent will typically be a group of formula **--NR--*,
wherein R is H, a C.sub.1-10 alkyl group, or an aryl group, wherein
* is the point of attachment of the amine group to the contrast
agent component, and wherein ** is the point of attachment of the
amine group to the group of formula (II). Typically some or all of
the amine groups of the contrast agent component are bound to a
group of formula (II). Usually, some but not all of the amine
groups of the contrast agent component are bound to a group of
formula (II).
[0111] An alcohol of the contrast agent component is usually an
group HO--*, wherein * is the point of attachment of the amine
group to the contrast agent component. When the group of formula
(II) is bonded to the alcohol group by Z, the hydrogen of the
alcohol group is typically replaced by Z. Therefore, when the group
of formula (II) is bonded to the contrast agent component by Z, the
alcohol group of the contrast agent will typically be a group of
formula **--O--*, wherein * is the point of attachment of the
alcohol group to the contrast agent component, and wherein ** is
the point of attachment of the alcohol group to the group of
formula (II). Typically some or all of the alcohol groups of the
contrast agent component are bound to a group of formula (II).
Usually, some but not all of the alcohol groups of the contrast
agent component are bound to a group of formula (II).
[0112] A carboxyl group of the contrast agent component is usually
a group HOC(O)--*, wherein * is the point of attachment of the
carboxyl group to the contrast agent component. When the group of
formula (II) is bonded to the carboxyl group by Z, the hydrogen of
the carboxyl group is typically replaced by Z. Therefore, when the
group of formula (II) is bonded to the contrast agent component by
Z, the carboxyl group of the contrast agent will typically be a
group of formula **--OC(O)--*, wherein * is the point of attachment
of the carboxyl group to the contrast agent component, and wherein
** is the point of attachment of the carboxyl group to the group of
formula (II). Typically some or all of the carboxyl groups of the
contrast agent component are bound to a group of formula (II).
Usually, some but not all of the carboxyl groups of the contrast
agent component are bound to a group of formula (II).
[0113] The functional group is typically an amine group. Usually,
therefore, the functional group is an amine group and Z is a bond
attaching the group of formula (II) to the nitrogen atom of the
amine group.
[0114] The above-mentioned conjugate typically comprises a
metal-containing nanoparticle. The nanoparticle in the conjugate of
the invention typically comprises (a) a metal or metal compound and
(b) a biocompatible coating. Usually, the biocompatible coating
comprises a plurality of functional groups. Typically, the or each
linker group described above is bonded to a functional group.
Often, therefore, the conjugate comprises metal-containing
particles, which comprise (a) a metal or metal compound and (b) a
biocompatible coating, wherein Z in formula (II) is a bond
attaching the group of formula (II) to a functional group. The
functional groups are often amine, alcohol or carboxylic acid
groups, as discussed above. It is usually the case that the
functional groups are amine groups, in which case Z is typically a
bond attaching the group of formula (II) to the nitrogen atom of
the amine group. The above-mentioned conjugate may therefore
comprise a plurality of hypoxia targeting moieties, each of which
is covalently bonded to the MRI contrast agent component by a
linker group of formula (II), wherein Z in each linker group is a
bond attaching the group of formula (II) to the nitrogen atom of an
amine group.
[0115] The nanoparticle is usually at least partially covered with
the biocompatible coating. For example, at least 10% of the surface
of the nanoparticle may be covered with the biocompatible coating.
Often, at least 30% of the surface of the nanoparticle is covered
with the biocompatible coating. For instance, at least 50% of the
surface of the nanoparticle may be covered, or for example at least
75% of the surface such as at least 90% of the surface may be
covered. For instance, the nanoparticle may be completely covered
by the biocompatible coating.
[0116] The number of hypoxia targeting moieties which may be
conjugated to the MRI contrast component can be assessed in terms
of absolute numbers. For example, the MRI contrast component may be
conjugated to more than 100 hypoxia targeting moieties, or for
instance to more than 1000 hypoxia targeting moieties. It may for
instance be conjugated to more than 10,000 hypoxia targeting
moieties, or for instance to more than 100,000 hypoxia targeting
moieties.
[0117] Alternatively, the number of hypoxia targeting moieties
which may be conjugated to the MRI contrast component may be
defined in terms of a percentage covering. For example, this may
refer to the number of potential binding sites for the hypoxia
targeting moiety on the surface of the MRI contrast component.
Typically, from 1% to 99% of such sites are conjugated to hypoxia
targeting moieties, such as from 10% to 90%, more usually from 20%
to 80% such as from 30% to 70%. Usually at least 10% of such sites
are conjugated to hypoxia targeting moieties, for instance at least
30%, such as for example at least 50% or at least 60%, for instance
at least 70%.
[0118] When the nanoparticle comprises a biocompatible coating
which comprises amine groups, the said binding sites are typically
the amine groups. At least 10% of the amine groups of the
biocompatible coating may be attached to a group of formula (II).
Often, for instance, at least 30% of the amine groups of the
biocompatible coating are attached to a group of formula (II). For
instance, at least 50% or at least 60%, for example at least 70% of
the amine groups of the biocompatible coating may be attached to a
group of formula (II). When the nanoparticle comprises a
biocompatible coating which comprises alcohol or carboxyl groups,
the said binding sites are typically the alcohol or carboxyl
groups. At least 10%, often at least 30%, for instance at least 50%
or at least 60%, such as at least 70% of the alcohol or carboxyl
groups of the biocompatible coating may be attached to a group of
formula (II).
[0119] As discussed above, the amine groups which are attached to a
group of formula (II) will typically have the structure **--NR--*,
wherein * is the point of attachment of the amine group to the
nanoparticle coating, and ** is the point of attachment of the
amine group to the group of formula (II), and R is H, C.sub.1-10
alkyl or aryl. Often, R is H or C.sub.1-6 alkyl. Usually R is H.
The other amine groups in the conjugate which are not attached to a
group of formula (II) will typically have the corresponding
structure H(R)N--* wherein R and * are as defined above. Similarly,
the alcohol groups which are attached to a group of formula (II)
will typically have the structure **--O--*, wherein * is the point
of attachment of the alcohol group to the nanoparticle coating, and
** is the point of attachment of the alcohol group to the group of
formula (II). The other alcohol groups in the conjugate which are
not attached to a group of formula (II) will typically have the
corresponding structure HO--* wherein * is as defined above.
Likewise, the carboxyl groups which are attached to a group of
formula (II) will typically have the structure **--OC(O)--*,
wherein * is the point of attachment of the carboxyl group to the
nanoparticle coating, and ** is the point of attachment of the
carboxyl group to the group of formula (II). The other carboxyl
groups in the conjugate which are not attached to a group of
formula (II) will typically have the corresponding structure
HOC(O)--* wherein * is as defined above.
[0120] As defined herein, the conjugate comprises a nanoparticle
which may comprise a biocompatible coating. The biocompatible
coating may comprise materials such as carbohydrates, sugars
(including long-chain sugars and the like), sugar alcohols,
poly(ethylene glycols (PEGs), nucleic acids, amino acids, peptides,
lipids and the like. For example, the biocompatible coating may
comprise dextran, carbodextran, mannan, cellulose or starch-based
polymers. It is also possible to use materials such as dendrimers.
Usually, the coating comprises dextran. The coating may for
instance consist of dextran. Where the biocompatible coating
comprises materials that are cross-linkable, the coating may be
cross-linked; alternatively the coating may not be cross-linked.
The biocompatible coating often, for example, comprises
cross-linked dextran.
[0121] The biocompatible coating may be functionalised by any
method known to those skilled in the art. For example, the
biocompatible coating may be functionalised by oxidation,
reduction, ligation, conjugation, or by reaction with further
chemical moieties. In such a manner, a wide range of functional
groups can be introduced into the biocompatible coating. The
biocompatible coating may either provide or can be pre-reacted in
order to provide functionalisation capable of bonding to the linker
groups. For example, a dextran coating can provide amine groups
which are capable of being reacted with a linker group in a process
for preparing the nanoparticles. Alternatively, a coating can be
pre-reacted, prior to reaction with a linker group, to form a
functional group which is capable of reacting with a linker group
in a process for preparing the nanoparticles.
[0122] In the case where the biocompatible coating naturally
contains functional groups, these functional groups may be used
without further modification. Alternatively, the functional groups
may be modified, or additional functional groups may be introduced.
Useful functional groups include amines, alcohols, carboxylic
acids, esters (including activated esters), epoxides, and the like.
Functional groups often employed include amines, alcohols, and
carboxylic acids; and amines are typically used. Examples of
structures of these kinds of groups, both when they are, and when
they are not, bonded to a linker group, are discussed above. Thus,
the biocompatible coating may comprise amine-functionalised
dextran, which amine-functionalised dextran may comprise a
plurality of amine groups. The biocompatible coating may be
obtained by any method known to those skilled in the art. For
instance, the biocompatible coating may comprises
amine-functionalised dextran which is obtainable by treating
dextran with epichlorohydrin and ammonia or ammonium hydroxide.
[0123] The MRI contrast agent component comprises a nanoparticle,
which nanoparticle comprises a metal or a metal compound. The metal
or metal compound may comprise any metal or metal compound that can
be used in MRI applications. Suitable metal or metal compounds
include those which are paramagnetic. Often, the metal or metal
compound is ferromagnetic or ferrimagnetic. Usually, the metal or
metal compound is superparamagnetic. Typically, the metal or metal
compound is capable of accelerating the dephasing of protons in an
MRI experiment by shortening the T.sub.2 and T.sub.2* relaxation
times.
[0124] The metal or metal compound may for instance comprise iron,
gadolinium, manganese, cobalt or nickel, or an alloy comprising
iron, gadolinium, manganese, cobalt or nickel. For instance, the
nanoparticle may comprise a metal compound which is an oxide of
iron or a mixed oxide of iron and another metal, or an oxide of
chromium. Usually, however, the metal or metal compound comprises
iron. The metal compound may for instance be iron oxide. The iron
oxide typically comprises Fe.sub.2O.sub.3 or Fe.sub.3O.sub.4.
Often, the metal compound comprises a mixed oxide of iron and
another metal such as for instance a mixed oxide of (a) iron and
(b) a second metal selected from cobalt, nickel, manganese,
beryllium, magnesium, calcium, barium, strontium, copper, zinc,
platinum, aluminium, chromium, bismuth, and a rare earth metals.
Sometimes, the iron may be present as an iron-platinum
nanoparticle.
[0125] The nanoparticles used in the MRI contrast agent component
can be made by any suitable method known in the art as may be
identified by the skilled person. For example, nanoparticles may be
made by attrition, where macro- or micro-scale particles are ground
in a mill, such as a planetary ball mill. Nanoparticles may also be
made by pyrolysis, wherein a vaporous precursor is forced through
an orifice at high pressure and burned, with the resulting solids
comprising oxide particles. Alternatively, a thermal plasma can be
used to vaporize micrometer-size particles, or an radio frequency
(RF) induction plasma torch can be used. In some aspects, inert-gas
condensation can be used to make nanoparticles from metals with low
melting points. Nanoparticles can alternatively be formed using
radiation chemistry. In a preferred method, nanoparticles are
derived from metal salts in solution, typically under anaerobic
condutions. For example, iron oxide nanoparticles may be derived by
co-precipitation from iron chloride hydrates in aqueous solution.
Many such nanoparticles, including iron oxide nanoparticles, are
also commercially available.
[0126] In one embodiment, the invention provides a conjugate
wherein: [0127] the nanoparticle comprises (a) iron oxide, and (b)
a biocompatible coating comprising amine-functionalised dextran,
which amine-functionalised dextran comprises a plurality of amine
groups; and [0128] the conjugate comprises a plurality of hypoxia
targeting moieties, each of which is a 2-nitroimidazolyl group, and
each of which is covalently bonded, via a linker group, to the
nitrogen atom of a different one of amine groups of the
amine-functionalised dextran, [0129] wherein each hypoxia targeting
moiety and linker group together form a group of formula (III)
##STR00005##
[0130] wherein * is the point of attachment of the group of formula
(III) to the nitrogen atom of one of the said amine groups.
Typically, at least 10% of the amine groups of the biocompatible
coating are attached to a group of formula (III). Often, for
instance, at least 30% of the amine groups are attached to a group
of formula (III). Thus, at least 50%, at least 60% or at least 70%
of the amine groups may be attached to a group of formula
(III).
[0131] The conjugates of the invention are useful in medical
applications. Thus, the present invention therefore provides, in
addition to a conjugate as defined above, a composition comprising
a conjugate of the invention and a pharmaceutically acceptable
excipient. For the avoidance of doubt, the conjugate of the
invention can, if desired, be used in the form of a solvate. The
composition of the invention comprises a conjugate of the
invention, or a pharmaceutically acceptable salt thereof, and a
pharmaceutically acceptable carrier or diluent. A composition of
the invention typically contains up to 85 wt % of a conjugate of
the invention. More typically, it contains up to 50 wt % of a said
conjugate. Preferred pharmaceutical compositions are sterile and
pyrogen free. Further, the pharmaceutical compositions provided by
the invention typically contain a compound of the invention which
is a substantially pure optical isomer.
[0132] The present invention further provides a contrast agent
which comprises a conjugate of the invention or a composition of
the invention. The contrast agent finds use as, for example, an MRI
contrast agent.
[0133] Either one of the conjugate of the invention and the
composition of the invention can be used as a contrast agent.
Typically, such use is use as a contrast agent for use in magnetic
resonance (MR) applications such as magnetic resonance imaging
(MRI). Thus, the conjugate of the invention or the composition of
the invention can be used as a MRI contrast agent. For example, the
conjugate of the invention or the composition of the invention can
be used as a contrast agent for detecting hypoxia.
[0134] The conjugate of the invention finds use in a method of
imaging a subject. Thus, the invention provides a method of imaging
a subject, which method comprises: (a) administering to the subject
a conjugate of the invention, a composition of the invention or a
contrast agent of the invention; and (b) imaging the subject.
Typically, the method comprises (b) imaging the subject by MRI.
[0135] The invention also provides a method of detecting hypoxia in
a subject, which method comprises: (a) administering to the subject
a conjugate of the invention, a composition of the invention, or a
contrast agent of the invention; and (b) detecting the conjugate in
the subject by MRI.
[0136] The detecting of the conjugate by MRI may comprise imaging
the myocardium. Such a method is particularly useful when the
subject (i) has suffered from or is suffering from cardiac arrest,
or (ii) is susceptible to cardiac arrest.
[0137] The detecting of the conjugate by MRI may comprise imaging a
cancerous, pre-cancerous or benign tumour or growth. Such a method
is particularly valuable when the subject (i) has suffered from or
is suffering from cancer, or (ii) is susceptible to cancer. The
type of cancer that may be imaged by the method is not particularly
limited. The conjugates of the invention are suitable for imaging
any kind of cancer that can be imaged by MRI. For example, the
cancer may be lung cancer, prostate cancer, colorectal cancer, and
stomach cancer, breast cancer, or cervical cancer. In some cases
skin cancer may also be imaged.
[0138] Other cancers include acute lymphoblastic leukaemia, brain
tumors and non-Hodgkin lymphoma. In other examples, the cancer may
be a cancer of the appendix, bladder, brain, central nervous system
(CNS), colon, gall bladder, heart, kidney, liver, mouth, ovary,
pancreas, small or large intestine, testes, throat, thyroid, or
uterus. The cancer may be caused by leukemia or lymphoma.
[0139] In one aspect, the subject is a mammal, in particular a
human. However, it may be non-human. Non-human animals include, but
are not limited to, primates, such as marmosets or monkeys,
commercially farmed animals, such as horses, cows, sheep or pigs,
and pets, such as dogs, cats, mice, rats, guinea pigs, ferrets,
gerbils or hamsters. The subject can be any animal that is capable
of experiencing hypoxia.
[0140] The invention also provides an in vitro method of imaging a
cell or tissue sample, which method comprises: (a) contacting the
cell or tissue sample with a conjugate of the invention, a
composition of the invention, or a contrast agent of the invention;
and (b) imaging the cell or tissue sample. Usually, the method
comprises (b) imaging the cell or tissue sample by MRI.
[0141] As mentioned previously, the conjugates of the invention
find use in medical methods. Thus, provided is a conjugate of the
invention, a composition of the invention, or a contrast agent of
the invention, for use in a diagnostic method practised on the
human or animal body. The diagnostic method may be a diagnostic
method practised on the human or animal body for diagnosing a
disease or condition associated with hypoxia.
[0142] The disease or condition associated with hypoxia is not
particularly limited, and may be any disease that is characterised
by altering or increasing hypoxia in the human or animal body. For
example, the disease may be cancer (exemplary cancers are provided
above). Alternatively, the disease or condition may be a disease or
condition associated with cardiac arrest. The disease or condition
that is associated with cardiac arrest is not particularly limited,
and may include ventricular fibrillation, coronary heart disease,
cardiomyopathy, congenital heart disease, heart valve disease,
acute myocarditis or Long QT Syndrome. The disease or condition may
be a non-ischemic heart disease (including cardiac rhythm
disturbances, hypertensive heart disease and congestive heart
failure). Alternatively, the disease or condition may be caused by
coronary artery abnormalities, myocarditis, or hypertrophic
cardiomyopathy. The disease or condition that is associated with
cardiac arrest may be unrelated to a heart problem. For example,
the disease or condition may arise from trauma, non-trauma related
bleeding (such as gastrointestinal bleeding, aortic rupture, and
intracranial haemorrhage), overdose, drowning or pulmonary
embolism. Environmental toxins from for example certain jellyfish
may also cause cardiac arrest.
[0143] From the discussion provided herein, it will thus be clear
to the skilled person that the invention provides a conjugate as
defined herein, a composition as defined herein, or a contrast
agent as defined herein, for use in a method of imaging a subject
or a method of detecting hypoxia in a subject, as defined
herein.
[0144] The invention further provides a method of evaluating the
activity of a pharmaceutical, which method comprises: [0145] (i)
administering to a subject a conjugate of the invention, a
composition of the invention, or a contrast agent of the invention;
[0146] (ii) detecting the conjugate in the subject by MRI prior to
administering the pharmaceutical to the subject; [0147] (iii)
administering the pharmaceutical to the subject; [0148] (iv)
detecting the conjugate in the subject by MRI after administering
the pharmaceutical to the subject; and [0149] (v) evaluating
changes in the MRI response of the conjugate before and after
administration of the pharmaceutical.
[0150] For instance, the pharmaceutical may be for the treatment,
prevention or suppression of a disease or condition associated with
hypoxia, including the conditions associated with hypoxia
identified above. For example, the pharmaceutical may be for the
treatment, prevention or suppression of a disease or condition such
as cancer, ventricular fibrillation, coronary heart disease,
cardiomyopathy, congenital heart disease, heart valve disease,
acute myocarditis or Long QT Syndrome. For instance, the subject
may be an animal model for cardiac arrest or cancer.
[0151] As discussed herein, the conjugate of the invention
(including pharmaceutically acceptable salts thereof), the
composition of the invention and the contrast agent of the
invention are medically useful, for example in diagnostic methods
as described herein. The conjugate of the invention (including
pharmaceutically acceptable salts thereof), the composition of the
invention or the contrast agent of the invention can be
administered to the subject in circumstances wherein the subject
can be asymptomatic. The subject is typically one that is at risk
of cardiac arrest or cancer, or one that has previously experienced
cardiac arrest or cancer. Alternatively, the conjugate of the
invention (including pharmaceutically acceptable salts thereof),
the composition of the invention or the contrast agent of the
invention can be administered to the subject in circumstances
wherein the subject can be symptomatic. The subject is typically
one that is suffering from cardiac arrest or cancer.
[0152] The conjugate of the invention (including pharmaceutically
acceptable salts thereof), the composition of the invention or the
contrast agent of the invention may be administered in a variety of
dosage forms. Thus, it can be administered orally, for example as
tablets, troches, lozenges, aqueous or oily suspensions,
dispersible powders or granules. The conjugate of the invention
(including pharmaceutically acceptable salts thereof), the
composition of the invention or the contrast agent of the invention
may also be administered parenterally, whether subcutaneously,
intravenously, intramuscularly, intrasternally, transdermally or by
infusion techniques. They may also be administered as a
suppository.
[0153] The conjugate of the invention (including pharmaceutically
acceptable salts thereof), the composition of the invention or the
contrast agent of the invention is typically formulated for
administration with a pharmaceutically acceptable carrier or
diluent. For example, solid oral forms may contain, together with
the active compound, diluents, e.g. lactose, dextrose, saccharose,
cellulose, corn starch or potato starch; lubricants, e.g. silica,
talc, stearic acid, magnesium or calcium stearate, and/or
polyethylene glycols; binding agents; e.g. starches, arabic gums,
gelatin, methylcellulose, carboxymethylcellulose or polyvinyl
pyrrolidone; disaggregating agents, e.g. starch, alginic acid,
alginates or sodium starch glycolate; effervescing mixtures;
dyestuffs; sweeteners; wetting agents, such as lecithin,
polysorbates, laurylsulphates; and, in general, non toxic and
pharmacologically inactive substances used in pharmaceutical
formulations. Such pharmaceutical preparations may be manufactured
in known manner, for example, by means of mixing, granulating,
tableting, sugar coating, or film coating processes.
[0154] Liquid dispersions for oral administration may be syrups,
emulsions and suspensions. The syrups may contain as carriers, for
example, saccharose or saccharose with glycerine and/or mannitol
and/or sorbitol.
[0155] Suspensions and emulsions may contain as carrier, for
example a natural gum, agar, sodium alginate, pectin,
methylcellulose, carboxymethylcellulose, or polyvinyl alcohol. The
suspension or solutions for intramuscular injections may contain,
together with the active compound, a pharmaceutically acceptable
carrier, e.g. sterile water, olive oil, ethyl oleate, glycols, e.g.
propylene glycol, and if desired, a suitable amount of lidocaine
hydrochloride.
[0156] Solutions for injection or infusion may contain as carrier,
for example, sterile water or, usually, they may be in the form of
sterile, aqueous, isotonic saline solutions. Pharmaceutical
compositions suitable for delivery by needleless injection, for
example, transdermally, may also be used.
[0157] The dose of the conjugate of the invention (including
pharmaceutically acceptable salts thereof), the composition of the
invention or the contrast agent of the invention which is
administered to the subject may be determined according to various
parameters, especially according to the compound used; the age,
weight and condition of the subject to be treated; the route of
administration; and the required regimen. A physician will be able
to determine the required route of administration and dosage for
any particular subject. A typical daily dose is from about 0.01 to
100 mg per kg, usually from about 0.1 mg/kg to 50 mg/kg, e.g. from
about 1 to 10 mg/kg of body weight, according to the activity of
the specific conjugate, composition or contrast agent, the age,
weight and conditions of the subject to be treated, the type and
severity of the disease or condition and the frequency and route of
administration. Typically, daily dosage levels are from 5 mg to 2
g.
[0158] The conjugate of the invention (including pharmaceutically
acceptable salts thereof), the composition of the invention or the
contrast agent of the invention may further comprise instructions
to enable the kit to be used in the methods described herein or
details regarding which subjects the method may be used for.
Synthesis
[0159] The conjugates described herein can be produced by any
suitable method known in the art. Many such methods will occur to
the skilled person. For example, a nanoparticle may be modified
with a biocompatible coating, the coating may be modified with a
linker and then the linker-coated nanoparticle composite may be
reacted with a hypoxia targeting moiety.
[0160] Alternatively, the linker and hypoxia targeting moiety may
be pre-reacted, and the nanoparticle may be pre-coated with the
biocompatible coating, before the coated nanoparticle is reacted
with the linker-hypoxia targeting moiety composite. Alternatively,
the hypoxia targeting moiety may be pre-reacted with the linker,
which composite thus formed may then be reacted with the
biocompatible coating, before the linker-hypoxia targeting
moiety-coating composite is reacted with the nanoparticle. Any
method which is suitable for resulting in the conjugates of the
invention may be used. For example, the nanoparticle may be
synthesized and then coated with the biocompatible coating.
Separately, the linker may be synthesized and reacted with the
hypoxia targeting moiety. Subsequently, the coated nanoparticle may
be reacted with the linker-hypoxia targeting moiety composite to
yield the conjugate of the invention. Any suitable synthetic route
may be used to obtain the conjugates of the invention, and many
suitable reaction conditions may occur to the skilled person. For
example, when the hypoxia targeting moiety is a heteroaryl group,
(e.g. a nitroimidazolyl group), a skilled organic chemist can
easily attach a linker group to the ring using known chemistry
(e.g. by reacting a halide-functionalised linker to an NH group in
the heteroaryl ring). The attached linker can then be
functionalised with a group, such as for instance an C(.dbd.X)OR
group, where X is O or NH, and R is H or C.sub.1-6 alkyl, that is
then able to react in a coupling reaction with functional groups
(such as for instance --NHR groups) in a coating on the
nanoparticle. Many coupling reactions are known to the skilled
person, and many pairs of complementary functional groups that can
react together in a coupling reaction are known. Any suitable pair
of complementary functional groups can be used. Examples of pairs
of complementary functional groups that can react together in a
coupling reaction include the reaction of an --NHR group with a
C(.dbd.X)OR group (for instance the reaction of an --NH.sub.2 group
with a --C(NH)OMe group, or the reaction between an --NH.sub.2
group and a --COOH group), the reaction of an azide group with an
alkyne group, and the [4+2] cycloaddition of a diene with a
dienophile.
[0161] The following Examples illustrate the invention. They do not
however, limit the invention in any way. In this regard, it is
important to understand that the particular assays used in the
Examples section are designed only to provide an indication of the
suitability of the conjugates thus described for use in MR imaging
technologies. There are many assays available to determine such
suitability, and a negative result in any one particular assay is
therefore not determinative.
EXAMPLES
Experimental
[0162] Dynamic light scattering (DLS) and Zeta-Potential (ZP) were
measured on a DynaPro Titan, Wyatt Technology Corporation (laser
wavelength 830 nm, scattering angle 20.degree.) with Dynamics
software Version 6.9.2.11 or Malvern Instruments Zetsizer NanoZS90
instrument equipped with a 633 nm laser at a fixed scattering angle
of 173.degree. with Malvern Zetasizer software 6.32. All elemental
analysis data (determination of C, H, N and Fe) was carried out by
MEDAC Ltd. All nuclear magnetic resonance (NMR) spectroscopy was
carried out on a Bruker AVG 400. Chemical shifts are quoted as
.delta.-values and referenced to residual solvent. Multiplicities
so far were abbreviated to the following: s, singlet; d, doublet;
t, triplet; q, quartet; m, multiplet. Low resolution mass spectra
(MS) were recorded by positive or negative ion electrospray on a
Waters 2777 Sample Manager. Infrared spectra were recorded on a
Bruker Tensor 27 FT-IR spectrophotometer. Thin layer chromatography
(TLC) was performed on Merck Kieselgel 60F254 pre-coated aluminium
backed plates and spots detection was achieved using an ultraviolet
lamp (.lamda..sub.max=254 nm). Purifications by flash column
chromatography were carried out using Sorbsil C60 40/60 silica. All
water used in the Examples was, unless otherwise stated, purified
to a resistivity of 18.2 M.OMEGA.cm (Milli-Q, Millipore).
Iron Content
[0163] It is of importance to know the exact iron mass in the
suspension for magnetic tests purposes. Spectroscopic measurements
are most easily performed with liquid samples. To convert the
insoluble Fe.sub.3O.sub.4 in nanoparticles to a soluble species, an
acid digestion was performed to yield a solution of Fe.sup.3+. Iron
content measurements were obtained from a calibration curve
generated for known standards. To generate the calibration curve,
10 .mu.l of aqueous 3% freshly prepared H.sub.2O.sub.2 solution and
10 .mu.l of each standard solution was added to 1 ml of 5 M HCl. A
typical calibration curve is shown in FIG. 1, and depicts the
relationship between iron atomic absorbance (y-axis) and the Fe
concentration (x-axis) (mg/ml). The nanoparticles to be tested were
prepared by taking the ratio of 1/3 of nanoparticles solution to
2/3 water (Millipore), in triplicate. 2 .mu.l of the nanoparticles
from the dilution were incubated with 2 .mu.l of aqueous 3% freshly
prepared H.sub.2O.sub.2 solution and 200 .mu.l of 5 M HCl. The
nanoparticles were incubated at 50.degree. C. for 1 hour in a
thermo (heat) block. The standard curve and the sample absorbance
at 410 nm were measured on 96 well plate in triplicate. The
absorbance was subtracted from the background, and the standard
curve was plotted. The iron was calculated from the slope of the
standard curve generated. All the iron content measurement were
calculated based on the generated standard curve below.
Nanoparticles Characterization
[0164] Nanoparticles were precipitated by centrifugation. The
magnetic properties of the nanoparticles allowed their facile
removal, washing and isolation from a reaction solution using a
magnet held against the side of the container in which the
nanoparticles were located.
Dynamic Light Scattering (DLS)
[0165] DLS analysis allows the average size of a batch of
nanoparticles to be determined. Particles at a concentration of
0.4-0.5 mg.sup.-1 cm.sup.-1 (500 .mu.l) were dispersed in water
(Millipore, 18.2 M.OMEGA.cm). Nanoparticles were filtered through
0.1 or 0.4 micron filters (Millipore) unless otherwise stated. The
reported values are the average of five independent measurements;
each single measurement presents an average of ten measurements.
Data were recorded at 25.degree. C.
Zeta Potential (ZP)
[0166] Most particles in a colloidal system have a charged surface
due to proton or ionisable surface groups. These charged surfaces
give rise to an electric double layer comprising counter ions which
follow the particle motion. This layer of closely associated ions
is known as the Stern layer and the potential at this plane is
defined as the zeta potential. The electrophoretic mobility of the
nanoparticles can thus be determined by applying an external
current. Zeta Potential (ZP) measurements were conducted as
follows. 1 ml of 0.5 mg ml.sup.-1 particles was suspended in 10 mM
of sodium phosphate buffer (pH 7.0), MES
(2-(N-morpholino)ethanesulfonic acid, pH 7.2) or PBS (phosphate
buffered saline, pH 7.0). The buffer was filtrated on 0.1 .mu.m
prior to use in order to avoid multiscattering events. Zeta cells
were equilibrated at 21.degree. C. before recording three
measurements each of 12 runs. The data were fitted using the
Smoluchowski approximation assuming a Henry's function f(K.sub.a)
of 1.5. The electrophoretic mobility is converted to the zeta
potential using the Henry equation.
Example 1: Synthesis of Iron Oxide Nanoparticles (IONP)
[0167] Dextran (Pharmacosmos, product HX4271) (7.138 g, 0.649-0.793
mmol, 0.22-0.26 equivalent) was dissolved in Milli-Q water (20 mL)
on a rotary mixer for 20 minutes at an ambient temperature.
FeCl.sub.3.6H.sub.2O (1.351 g, 5.000 mmol, 1.67 equivalents) was
added to the dextran solution. The solution was then transferred to
a 250 mL round flask and deoxygenated under argon while stirring
(300 rpm) by repeated cycles of vacuum (100 mBar, 5 min) assisted
by sonication (degassing mode) followed by argon flushing for 5
minutes each cycle. After the first deoxygenation cycle,
FeCl.sub.2.4H.sub.2O (596/mg, 3.00 mmol, 1.00 equivalents) freshly
prepared and dissolved in water (Milli-Q, 5 mL) was added by
injecting through the rubber stoppers, and the solution was
deoxygenated by 4 more cycles. NH.sub.4OH (25%) (4.00 mL, 53.5
mmol, 17.8 equivalent) was introduced to the mixture at 142.9 mL/h
flow rate under vigorously mechanical stirring (600 rpm). The
reaction was then heated to 80.degree. C. in a water bath (15 min
from 24.degree. C. to 80.degree. C.) for 1 hour. The solution was
cooled (5 min at 0.degree. C. on ice) and dialyzed against 4 litres
of water (Millipore) through a SpectraPor dialysis membrane (MWCO
100 kDa) for 17 h to eliminate OH.sup.-, Cl.sup.-, NO.sub.3.sup.-,
SO.sub.4.sup.2-, CO.sub.3.sup.2-, NH.sub.4.sup.+, K.sup.+ and
Na.sup.+. The dialysis solution was changed after 2 and 4 hours and
left to proceed at an ambient temperature while gently stirring for
17 hours. The particles were collected and stored on 4.degree. C.
on a sample roller in order to prevent sedimentation of
nanoparticles.
Example 2: Amine Terminated Dextran Coated Nanoparticles
[0168] 20 mL of dextran covered nanoparticles (10 mg Fe) were
placed into a 250 mL round flask equipped with an oval stirrer bar.
While the solution was stirred at 500 rpm, 36.7 mL of freshly
prepared NaOH (5M) was added at a rate of 168 mL/h. 20 mL of
epichlorohydrin was then added at a rate of 94 mL/h. The mixture
was stirred at 1000 rpm for 7 h and then 20 mL of NH.sub.4OH (25%)
was added at a rate of 168 mL/h. The mixture was stirred at 1000
rpm for 14 hours at an ambient temperature. The mixture was
dialysed against water through a dialysis membrane. The mixture was
stirred for an additional 15 h and was then dialyzed against water
through a SpectraPor membrane (100,000 Da) for 22 h and then the
sample was concentrated by ultrafiltration (100 kDa membrane) to
approximately 18 mg Fe/mL. DLS and Zeta potential measurements were
conducted with values shown in Table 1.
Example 3: Linker Synthesis
2-(2-nitroimidazol-1-yl) acetonitrile (2.1)
##STR00006##
[0170] A mixture of 2-nitroimidazole (0.3 g), chloroacetonitrile
and DIPEA (N,N-diisopropylethylamine) in acetonitrile was heated at
65.degree. C. for 7 hours. Evaporation of the volatiles under
reduced pressure gave a brownish oil residue which was subject to
high vacuum overnight to remove the excess volatiles. The product
was then purified with a Pet/EtOAc gradient. Silica was washed with
50 mL of a Pet/EtOAc/Et.sub.3N (100:100:2) to deactivate silica
gel, to prevent possible decomposition. The column was run with
Pet/EtOAc; 50 mL of 70%:30%, 50 mL of 50%:50%, 100 mL of 30%:70%
and 100 mL of 0%:100%. The corresponding spot appeared in fractions
11-17. The test tubes containing these fractions were added to a
weighed round-bottom flask and the solvent evaporated under reduced
pressure to yield 279 mg (70%) of a white solid.
[0171] .sup.1H NMR (400 MHz, MeOD) .delta.=7.63 (1H, s), 7.23 (1H,
s), 5.58 (2H, s), .sup.13C NMR; .delta.=36.9, 127.76, 126.75. FTIR
(cm.sup.-1): 2923.7, 2853.4, 2360.4, 1537.9, 1492.0, 1369.7,
1284.5, 1155.7, 1136.0, 1080.3, 928.1, 837.9, 780.1, 758.6. HR-MS:
Mw calculated=175.0226 [M+Na].sup.+, Mw found=175.0227
[M+Na].sup.+. MP=124-126.degree. C.
2-(2-nitroimidazol-1-yl) 2-imido-2-methoxy-ethyl (IME, 2.2)
##STR00007##
[0173] Attachment of amino groups (e.g. amino-functionalised
nanoparticles) requires an amine-reactive group such as the
IME-linker (2.2). Reaction of 2.1 to form 2.2 is successful from pH
8-10. The reaction was tested using 0.4 and 4.0 equivalents of
sodium methoxide. Reaction with 0.4 eq. NaOMe led to a reaction pH
of ca. pH 9. Therefore, to a 50 ml round bottomed flask under
vacuum, 2.1 (0.01 g) was added in argon and diluted in anhydrous
methanol (130 mM). 0.4 eq. NaOMe was added and reaction stirred for
6 h at room temperature, continuously monitored by TLC and MS.
Almost total starting material spot was consumed, and the product
spot was analyzed by MS.
[0174] .sup.1H NMR (400 MHz, MeOD) .delta.=7.50 (1H, s), 7.19 (1H,
s), 5.23 (2H, s), 3.73 (3H, s); Mw found: 185.1 [M+H].sup.+; 207.1
[M+Na].sup.+; 391.1 [2M+Na].sup.+
[0175] The efficiency of the reaction of 2.1 to form 2.2 was
calculated by lyophilizing ca. 15 mg product under argon, and
conducting proton NMR. This revealed 96% formation of 2.2 for 0.4
equivalents of sodium methoxide used in the reaction.
Example 4: Nanoparticle Modification
[0176] Amine-terminated magnetic particles (500 .mu.g) were washed
4 times with methanol (2 mL per wash) and after the last wash, left
in a minimum volume of methanol. 2.1 (0.02 g) was diluted in
anhydrous methanol (130 mM) in a 2 ml vacuumed eppendorf, under
argon. NaOMe (0.4 eq.) was added and reaction agitated on an
orbital shaker for 2 days at room temperature. The suspension was
continuously monitored by TLC. Almost total starting material spot
was consumed, and IME (2.2) spot detected. The pH was monitored and
remained at ca. pH 9 during the reaction. The IME (2.2) spot was
still visible, suggesting that not all of it attached to the
nanoparticles. The particles were washed with methanol (3.times.2
mL), water (3.times.2 mL) and PBS (3.times.2 mL) prior to storage
in PBS (2 mL, final concentration=220 .mu.g(Fe)/mL). DLS and Zeta
potential measurements were conducted with values shown in Table
1.
[0177] The synthetic route for the synthesis of 2.1 and 2.2 and
subsequent nanoparticle modification is shown in FIG. 3.
DLS and Zeta Potential Measurements
[0178] Amine functionalised dextran coated nanoparticles have a
high degree of electrical stability and a highly charged surface.
Following modification with the linker and hypoxia sensing moiety
to form the conjugate, both the zeta potential (ZP) and
DLS-determined size increased, indicating successful modification
(see Table 1).
TABLE-US-00001 TABLE 1 ZP/mV Size (DLS)/nm Amine-functionalised
nanoparticle 26.6 53.15 Conjugate 33.3 62.6
Fmoc Numbering
[0179] Fmoc (Fluorenylmethyloxycarhonyl) number determination is
also a quantification method of amine loading. Free amine groups
are reacted with Fmoc-Cl, followed by a residue removal which
absorbs at .lamda.=290-315 nm (UV), being another indirect
quantitative measurement. Therefore, to a suspension of
amino-modified nanoparticles and the conjugate, Fmoc-Cl and
[0180] DIPEA were added. The reaction mixture was stirred at room
temperature overnight. The particles were separated using a magnet,
and repeatedly washed with DMF. Particles were re-suspended in 500
.mu.l of cleavage mixture (DMSO:DMF:DBU, 25:24:1) (DMSO: dimethyl
sulfoxide; DMF: dimethyl formamide; DBU:
1,8-Diazabicyclo[5.4.0]undec-7-ene). After 2 hours, particles were
separated with a magnet. 10 .mu.L of the solution was withdrawn and
added to 1 mL of MeOH (methanol). A blank was measured using 10
.mu.L of the cleavage mixture and 1 mL of MeOH. A calibration curve
(FIG. 4) was built from cleavage of Fmoc-Glycine (same conditions).
The absorbance was measured at .lamda.=295 nm. Data are shown in
Table 2.
TABLE-US-00002 TABLE 2 sample conc (M) Abs a 1.1840E-04 1.260553 b
9.4720E-05 0.916939 c 7.8933E-05 0.732927 d 6.7657E-05 0.617422 e
5.9200E-05 0.55613 f 3.9467E-05 0.334115 g 2.9600E-05 0.26601 h
1.9733E-05 0.151513 i 1.4800E-05 0.104092 l 1.1840E-05 0.067799
[0181] Results are shown in Table 3, and revealed a modification
level of 32%.
TABLE-US-00003 TABLE 3 Abs (295 nm) Correction Blank -0.00013 --
Amino-functionalised nanoparticle 0.027947 0.027817 Conjugate
0.0018766 0.01876
Fluorescamine Assay
[0182] Fluorescamine is non-fluorescent, and it is only possible to
detect fluorescence when a product forms from reaction with the
amino groups. Modification results in a reduction of fluorescence
intensity with increased levels of modification (since the amino
groups from the nanoparticles have already reacted with the
IME-probe 2.2). Thus, with a gradient of equivalents of the IME
2.2, a clear inverse correlation is expected.
[0183] Determination of the Background:
[0184] Non-modified particles (10 .mu.L, 2.6 mgFe/mL) were
suspended in PBS (140 .mu.L, 10 mM, pH 7.4). DMSO (50 .mu.L) was
then added into the mixture and the plate was kept at rt for 10 min
before measuring the fluorescence intensity (.lamda..sub.Ex: 405
nm, .lamda..sub.Em.: 460 nm). The measurements lead to the
fluorescence intensity of the background (IntBkgd).
[0185] Determination of the Fluorescence Intensity of the Starting
Material (0% Conversion):
[0186] The non-modified particles (10 .mu.L, 2.6 mgFe/mL) were
suspended in PBS (140 .mu.L, 10 mM, pH 7.4). Fluorescamine (50
.mu.L in DMSO, 0.625 mg/mL) was then added into the mixture and
plate was kept at rt for 10 min before measuring the fluorescence
intensity (.lamda..sub.Ex: 405 nm, .lamda..sub.Em.: 460 nm). The
measurements led to the fluorescence intensity of the starting
material (IntSM).
[0187] Determination of Particle Loading:
[0188] 2-nitroimidazole modified particles (10 .mu.L, 2.6 mgFe/mL)
were suspended in PBS (140 .mu.L, 10 mM, pH 7.4). Fluorescamine (50
.mu.L in DMSO, 0.625 mg/mL) was then added into the mixture and
plate was kept at rt for 10 min before measuring the fluorescence
intensity (.lamda..sub.Ex: 405 nm, .lamda..sub.Em.: 460 nm). The
measurements were performed in triplicates. The average of the 3
experiments led to the fluorescence intensity of the product
(Intp).
[0189] The particle loading percentage was given by Equation 1.
Particle Loading=100-[((Intp/IntBkgd)/(INtsm/IntBkgd)).times.100]
[Eq. 1]
[0190] Results are shown in Table 4, and revealed a modification
level of 12.7%.
TABLE-US-00004 TABLE 4 Correction (350 nm, 460 nm) Blank 0
Amino-NP's 87856 Modified NP's 76710
[0191] Deviations are typically expected between values obtained
from the Fmoc-numbering assay and the Fluorescamine assay. The data
above suggests that amidine formation in the particles was between
10% and 30%.
Example 5: Hypoxia and Contrast Tests
[0192] Mouse fibroblasts (3T3-Tet "Off") cells were prepared in 4
traits, 3 replicates each. Well plates were prepared at a density
of 300,000 cells/well. Control wells were included in which either
or both of the contrast agent and the hypoxia conditions (described
below) were omitted.
[0193] After the cells adhered to the wells, contrast agent was
added (100 .mu.l in 2 ml media; 5 .mu.g/ml) and left for overnight
incubation at 37.degree. C., 95% O.sub.2, 5% CO.sub.2. After 18 hr,
for the "hypoxia" wells, the media were replaced by
ischaemic-mimetic buffer (125.00 mM NaCl, 8.00 mM KCl, 6.25 mM
NaHCO.sub.3, 1.20 mM KH.sub.2PO.sub.4, 1.25 mM MgSO.sub.4, 1.20 mM
CaCl.sub.2, 20.00 mM HEPES
[4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid], 5.00 mM Na
lactate, pH 6.6) in the presence of 5 .mu.g/ml contrast agent and
stored for 5 hr in a sealed incubator with a supply of 0.1% 02,
37.degree. C., equipped with an air-lock chamber (Ruskinn Invivo2
400). Cell cultures that were incubated under normoxic conditions
remained in the same incubator as used for overnight treatment with
the contrast agent. Normoxic media were at pH 7.4.
[0194] All media were filter-sterilized before use, and media for
hypoxic incubations were pre-equilibrated overnight at the required
02 concentration in filter-capped tissue culture flasks. For
normoxia (95% O.sub.2, 5% CO.sub.2, 37.degree. C.), Dulbecco's
Modified Eagle's Medium was used (product code D6546, Sigma
Aldrich, UK). The medium comprised 10% Fetal Bovine Serum (FBS),
and L-glutamine in addition to the antibiotics penicillin,
streptomycin, puromycin and geneticin.
[0195] After 5-hours all cells were washed with media followed by a
wash with warm PBS (phosphate buffered saline). Then, trypsin was
added and cells were detached from the wells, the cell suspensions
added into 1.5 ml Eppendorf tubes and centrifuged at 10,000 rpm for
5 min at room temperature in a benchtop centrifuge. The pellets
were washed once more in PBS and the above centrifugation was
repeated. The final supernatant was aspirated and the pellets were
embedded in agarose, in the original Eppendorfs, for MR
analysis.
[0196] For evaluation of % cell death due to hypoxia, a replicate
from the 6 wells per treatment was kept, the pellets generated as
above and diluted into trypan blue. Number of trypan blue-positive
(therefore, dead) cells were counted using a Neubauer
haemocytometer, and calculated as % dead/total cell number. The
results are shown in FIG. 2.
[0197] The results shown in FIG. 2 reveal that under normoxic
conditions, ca. 3% of cells died after 5 hours treatment under the
experimental conditions used. Addition of the conjugate had minimal
effect on cell viability, with ca. 2% cell death. Under hypoxic
conditions, cell death percentages increased in both the presence
and absence of the conjugate. The difference between the levels of
cell death under hypoxic conditions in the presence and absence of
the conjugate was not significant. Even under hypoxic conditions in
the presence of the conjugate, more than 90% of cells remained
alive.
[0198] Before cellular samples were subjected to MRI analysis, they
were assessed by eye (see FIG. 5). It could clearly be seen that
after washing the cells, significantly more contrast agent remained
on the hypoxia treatment sample (tube 3), demonstrating the
selective targeting of the conjugate to hypoxic regions.
[0199] Preliminary MRI confirmed this favourable scenario. 2D
gradient echo images (TE/TR=6/20 ms) obtained on cells with/without
contrast, which had/had not been subjected to hypoxia showed a
strong T2*-weighted contrast, with a clear negative contrast
enhancement in case of hypoxia+contrast treatment (FIG. 6).
* * * * *